Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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OVARIAN TUMOR SEQUENCES AND METHODS OF USE THEREFOR
TECHNICAL FIELD
The present invention relates generally to ovarian cancer therapy. The
invention is more specifically related to polypeptides comprising at least a
portion of an
ovarian carcinoma protein, and to polynucleotides encoding such polypeptides,
as well
as antibodies and immune system cells that specifically recognize such
polypeptides.
Such polypeptides, polynucleotides, antibodies and cells may be used in
vaccines and
pharmaceutical compositions for treatment of ovarian cancer.
1o BACKGROUND OF THE INVENTION
Ovarian cancer is a significant health problem for women in the United
States and throughout the world. , Although advances have been made in
detection and
therapy of this cancer, no vaccine or other universally successful method for
prevention
or treatment is currently available. Management of the disease currently
relies on a
combination of early diagnosis and aggressive treatment, which may include one
or
more of a variety of treatments such as surgery, radiotherapy, chemotherapy
and
hormone therapy. The course of treatment for a particular cancer is often
selected based
on a variety of prognostic parameters, including an analysis of specific tumor
markers.
However, the use of established markers often leads to a result that is
difficult to
2o interpret, and high mortality continues to be observed in many cancer
patients.
Immunotherapies have the potential to substantially improve cancer
treatment and survival. Such therapies may involve the generation or
enhancement of
an immune response to an ovarian carcinoma antigen. However, to date,
relatively few
ovarian carcinoma antigens are known and the generation of an immune response
against such antigens has not been shown to be therapeutically beneficial.
Accordingly, there is a need in the art for improved methods for
identifying ovarian tumor antigens and for using such antigens in the therapy
of ovarian
cancer. The present invention fulfills these needs and further provides other
related
advantages.
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SUMMARY OF THE INVENTION
Briefly stated, this invention provides compositions and methods for the
therapy of cancer, such as ovarian cancer. In one aspect, the present
invention provides
polypeptides comprising an immunogenic portion of an ovarian carcinoma
protein, or a
variant thereof that differs in one or more substitutions, deletions,
additions and/or
insertions such that the ability of the variant to react with ovarian
carcinoma protein-
specific antisera is not substantially diminished. Within certain embodiments,
the
ovarian carcinoma protein comprises a sequence that is encoded by a
polynucleotide
sequence selected from the group consisting of SEQ ID NOs:l, 2, S, 9, 10, 13,
16, 19,
l0 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78,
80-82, 84, 86, 89-
93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136,
137, 140,
143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185 and 193-199,
and
complements of such polynucleotides.
The present invention further provides polynucleotides that encode a
polypeptide as described above or a portion thereof, expression vectors
comprising such
polynucleotides and host cells transformed or transfected with such expression
vectors.
Within other aspects, the present invention provides pharmaceutical
compositions and vaccines. Pharmaceutical compositions may comprise a
physiologically acceptable carrier or excipient in combination with one or
more of: (i) a
2o polypeptide comprising an immunogenic portion of an ovarian carcinoma
protein, or a
variant thereof that differs in one or more substitutions, deletions,
additions and/or
insertions such that the ability of the variant to react with ovarian
carcinoma protein-
specific antisera is not substantially diminished, wherein the ovarian
carcinoma protein
comprises an amino acid sequence encoded by a polynucleotide that comprises a
sequence recited in any one of SEQ ID NOs:I-185 and 187-199; (ii) a
polynucleotide
encoding such a polypeptide; (iii) an antibody that specifically binds to such
a
polypeptide; (iv) an antigen-presenting cell that expresses such a polypeptide
and/or (v)
a T cell that specifically reacts with such a polypeptide. Vaccines may
comprise a non-
specific immune response enhancer in combination with one or more of: (i) a
polypeptide comprising ax immunogenic portion of an ovarian carcinoma protein,
or a
variant thereof that differs in one or more substitutions, deletions,
additions and/or
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insertions such that the ability of the variant to react with ovarian
carcinoma protein-
specific antisera is not substantially diminished, wherein the ovarian
carcinoma protein
comprises an amino acid sequence encoded by a polynucleotide that comprises a
sequence recited in any one of SEQ ID NOs:I-185 and 187-196, (ii) a
polynucleotide
encoding such a polypeptide; (iii) an anti-idiotypic antibody that is
specifically bound
by an antibody that specifically binds to such a polypeptide; (iv) an antigen-
presenting
cell that expresses such a polypeptide and/or (v) a T cell that specifically
reacts with
such a polypeptide. An exemplary polypeptide comprises an amino acid sequence
recited in SEQ ID N0:186.
l0 The present invention further provides, in other aspects, fusion proteins
that comprise at least one polypeptide as described above, as well as
polynucleotides
encoding such fusion proteins.
Within related aspects, pharmaceutical compositions comprising a fusion
protein or polynucleotide encoding a fusion protein in combination with a
physiologically acceptable carrier are provided.
Vaccines are further provided, within other aspects, comprising a fusion
protein or polynucleotide encoding a fusion protein in combination with a non-
specific
immune response enhancer.
Within further aspects, the present invention provides methods for
2o inhibiting the development of a cancer in a patient, comprising
administering to a
patient a pharmaceutical composition or vaccine as recited above.
The present invention further provides, within other aspects, methods for
stimulating and/or expanding T cells, comprising contacting T cells with (a) a
polypeptide comprising an immunogenic portion of an ovarian carcinoma protein,
or a
variant thereof that differs in one or more substitutions, deletions,
additions and/or
insertions such that the ability of the variant to react with ovarian
carcinoma protein-
specific antisera is not substantially diminished, wherein the ovarian
carcinoma protein
comprises an amino acid sequence encoded by a polynucleotide that comprises a
sequence recited in any one of SEQ ID NOs:I-185 and 187-199; (b) a
polynucleotide
encoding such a polypeptide and/or (c) an antigen presenting cell that
expresses such a
polypeptide under conditions and for a time sufficient to permit the
stimulation and/or
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expansion of T cells. Such polypeptide, polynucleotide and/or antigen
presenting
cells) may be present within a pharmaceutical composition or vaccine, for use
in
stimulating and/or expanding T cells in a mammal.
Within other aspects, the present invention provides methods for
inhibiting the development of ovarian cancer in a patient, comprising
administering to a
patient T cells prepared as described above.
Within further aspects, the present invention provides methods for
inhibiting the development of ovarian cancer in a patient, comprising the
steps of: (a)
incubating CD4+ and/or CD8+ T cells isolated from a patient with one or more
of: (i) a
t o polypeptide comprising an immunogenic portion of an ovarian carcinoma
protein, or a
variant thereof that differs in one or more substitutions, deletions,
additions and/or
insertions such that the ability of the variant to react with ovarian
carcinoma protein-
specific antisera is not substantially diminished, wherein the ovarian
carcinoma protein
comprises an amino acid sequence encoded by a polynucleotide that comprises a
is sequence recited in any one of SEQ ID NOs:I-185 and 187-199; (ii) a
polynucleotide
encoding such a polypeptide; or (iii) an antigen-presenting cell that
expresses such a
polypeptide; such that T cells proliferate; and (b) administering to the
patient an
effective amount of the proliferated T cells, and thereby inhibiting the
development of
ovarian cancer in the patient. The proliferated cells may be cloned prior to
2o administration to the patient.
These and other aspects of the present invention will become apparent
upon reference to the following detailed description and attached drawings.
All
references disclosed herein are hereby incorporated by reference in their
entirety as if
each was incorporated individually.
25 DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed generally to compositions and their use
in the therapy and diagnosis of cancer, particularly ovarian cancer. As
described further
below, illustrative compositions of the present invention include, but are not
restricted
to, polypeptides, particularly immunogenic polypeptides, polynucleotides
encoding
3o such polypeptides, antibodies and other binding agents, antigen presenting
cells (APCs)
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and immune system cells (e.g., T cells).
The practice of the present invention will employ, unless indicated
specifically to the contrary, conventional methods of virology, immunology,
microbiology, molecular biology and recombinant DNA techniques within the
skill of
the art, many of which are described below for the purpose of illustration.
Such
techniques are explained fully in the literature. See, e.g., Sambrook, et al.
Molecular
Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular
Cloning:
A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D.
Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid
Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and
Translation (B.
Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986);
Perbal,
A Practical Guide to Molecular Cloning (1984).
All publications, patents and patent applications cited herein, whether
supra or infra, are hereby incorporated by reference in their entirety.
As used in this specification and the appended claims, the singular forms
"a," "an" and "the" include plural references unless the content clearly
dictates
otherwise.
POLYPEPTIDE COMPOSITIONS
2o As used herein, the term "polypeptide" " is used in its conventional
meaning, i.e. as a sequence of amino acids. The polypeptides are not limited
to a
specific length of the product; thus, peptides, oligopeptides, and proteins
are included
within the definition of polypeptide, and such terms may be used
interchangeably herein
unless specifically indicated otherwise. This term also does not refer to or
exclude post-
expression modifications of the polypeptide, for example, glycosylations,
acetylations,
phosphorylations and the like, as well as other modifications known in the
art, both
naturally occurring and non-naturally occurring. A polypeptide may be an
entire
protein, or a subsequence thereof. Particular polypeptides of interest in the
context of
this invention are amino acid subsequences comprising epitopes, i.e. antigenic
3o determinants substantially responsible for the immunogenic properties of a
polypeptide
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and being capable of evoking an immune response.
Particularly illustrative polypeptides of the present invention comprise those
encoded
by a polynucleotide sequence set forth herein, or a sequence that hybridizes
under
moderately stringent conditions, or, alternatively, under highly stringent
conditions, to a
polynucleotide sequence set forth herein.
The polypeptides of the present invention are sometimes herein referred
to as ovarian tumor proteins or ovarian tumor polypeptides, as an indication
that their
identification has been based at least in part upon their increased levels of
expression in
ovarian tumor samples. Thus, a " ovarian tumor polypeptide" or "ovarian tumor
1 o protein," refers generally to a polypeptide sequence of the present
invention, or a
polynucleotide sequence encoding such a polypeptide, that is expressed in a
substantial
proportion of ovarian tumor samples, for example preferably greater than about
20%,
more preferably greater than about 30%, and most preferably greater than about
50% or
more of ovarian tumor samples tested, at a level that is at least two fold,
and preferably
at least five fold, greater than the level of expression in normal tissues, as
determined
using a representative assay provided herein. A ovarian tumor polypeptide
sequence of
the invention, based upon its increased level of expression in tumor cells,
has particular
utility both as a diagnostic marker as well as a therapeutic target, as
further described
below.
In certain preferred embodiments, the polypeptides of the invention are
immunogenic, i.e., they react detectably within an immunoassay (such as an
ELISA or
T-cell stimulation assay) with antisera and/or T-cells from a patient with
ovarian cancer.
Screening for immunogenic activity can be performed using techniques well
known to
the skilled artisan. For example, such screens can be performed using methods
such as
those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring
Harbor Laboratory, 1988. In one illustrative example, a polypeptide may be
immobilized on a solid support and contacted with patient sera to allow
binding of
antibodies within the sera to the immobilized polypeptide. Unbound sera may
then be
removed and bound antibodies detected using, for example,'z5I-labeled Protein
A.
3o As would be recognized by the skilled artisan, immunogenic portions of
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the polypeptides disclosed herein are also encompassed by the present
invention. An
"immunogenic portion," as used herein, is a fragment of an immunogenic
polypeptide
of the invention that itself is immunologically reactive (i. e., specifically
binds) with the
B-cells and/or T-cell surface antigen receptors that recognize the
polypeptide.
Immunogenic portions may generally be identified using well known techniques,
such
as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven
Press,
1993) and references cited therein. Such techniques include screening
polypeptides for
the ability to react with antigen-specific antibodies, antisera and/or T-cell
lines or
clones. As used herein, antisera and antibodies are "antigen-specific" if they
specifically bind to an antigen (i.e., they react with the protein in an ELISA
or other
immunoassay, and do not react detectably with unrelated proteins). Such
antisera and
antibodies may be prepared as described herein, and using well-known
techniques.
In one preferred embodiment, an immunogenic portion of a polypeptide
of the present invention is a portion that reacts with antisera and/or T-cells
at a level that
is not substantially less than the reactivity of the full-length polypeptide
(e.g., in an
ELISA and/or T-cell reactivity assay). Preferably, the level of immunogenic
activity of
the immunogenic portion is at least about 50%, preferably at least about 70%
and most
preferably greater than about 90% of the immunogenicity for the full-length
polypeptide. In some instances, preferred immunogenic portions will be
identified that
have a level of immunogenic activity greater than that of the corresponding
full-length
polypeptide, e.g., having greater than about 100% or 150% or more immunogenic
activity.
In certain other embodiments, illustrative immunogenic portions may
include peptides in which an N-terminal leader sequence and/or transmembrane
domain
have been deleted. Other illustrative immunogenic portions will contain a
small N-
and/or C-terminal deletion (e.g., 1-30 amino acids, preferably 5-15 amino
acids),
relative to the mature protein.
In another embodiment, a polypeptide composition of the invention may
also comprise one or more polypeptides that are immunologically reactive with
T cells
3o and/or antibodies generated against a polypeptide of the invention,
particularly a
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polypeptide having an amino acid sequence disclosed herein, or to an
immunogenic
fragment or variant thereof.
In another embodiment of the invention, polypeptides are provided that
comprise one or more polypeptides that are capable of eliciting T cells and/or
antibodies
that are immunologically reactive with one or more polypeptides described
herein, or
one or more polypeptides encoded by contiguous nucleic acid sequences
contained in
the polynucleotide sequences disclosed herein, or immunogenic fragments or
variants
thereof, or to one or more nucleic acid sequences which hybridize to one or
more of
these sequences under conditions of moderate to high stringency.
to The present invention, in another aspect, provides polypeptide fragments
comprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino
acids, or more,
including all intermediate lengths, of a polypeptide compositions encoded by a
polynucleotide sequence set forth herein.
In another aspect, the present invention provides variants of the
polypeptide compositions described herein. Polypeptide variants generally
encompassed by the present invention will typically exhibit at least about
70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity
(determined as described below), along its length, to a polypeptide sequences
set forth
herein.
2o In one preferred embodiment, the polypeptide fragments and variants
provide by the present invention are immunologically reactive with an antibody
and/or
T-cell that reacts with a full-length polypeptide specifically set for the
herein.
In another preferred embodiment, the polypeptide fragments and variants
provided by the present invention exhibit a level of immunogenic activity of
at least
about 50%, preferably at least about 70%, and most preferably at least about
90% or
more of that exhibited by a full-length polypeptide sequence specifically set
forth
herein.
A polypeptide "variant," as the term is used herein, is a polypeptide that
typically differs from a polypeptide specifically disclosed herein in one or
more
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substitutions, deletions, additions and/or insertions. Such variants may be
naturally
occurring or may be synthetically generated, for example, by modifying one or
more of
the above polypeptide sequences of the invention and evaluating their
immunogenic
activity as described herein and/or using any of a number of techniques well
known in
the art.
For example, certain illustrative variants of the polypeptides of the
invention include those in which one or more portions, such as an N-terminal
leader
sequence or transmembrane domain, have been removed. Other illustrative
variants
include variants in which a small portion (e.g., 1-30 amino acids, preferably
5-15 amino
1o acids) has been removed from the N- andlor C-terminal of the mature
protein.
In many instances, a variant will contain conservative substitutions. A
"conservative substitution" is one in which an amino acid is substituted for
another
amino acid that has similar properties, such that one skilled in the art of
peptide
chemistry would expect the secondary structure and hydropathic nature of the
polypeptide to be substantially unchanged. As described above, modifications
may be
made in the structure of the polynucleotides and polypeptides of the present
invention
and still obtain a functional molecule that encodes a variant or derivative
polypeptide
with desirable characteristics, e.g., with immunogenic characteristics. When
it is
desired to alter the amino acid sequence of a polypeptide to create an
equivalent, or
2o even an improved, immunogenic variant or portion of a polypeptide of the
invention,
one skilled in the art will typically change one or more of the codons of the
encoding
DNA sequence according to Table 1.
For example, certain amino acids may be substituted for other amino
acids in a protein structure without appreciable loss of interactive binding
capacity with
structures such as, for example, antigen-binding regions of antibodies or
binding sites
on substrate molecules. Since it is the interactive capacity and nature of a
protein that
defines that protein's biological functional activity, certain amino acid
sequence
substitutions can be made in a protein sequence, and, of course, its
underlying DNA
coding sequence, and nevertheless obtain a protein with like properties. It is
thus
3o contemplated that various changes may be made in the peptide sequences of
the
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disclosed compositions, or corresponding DNA sequences which encode said
peptides
without appreciable loss of their biological utility or activity.
TABLE 1
Amino Acids Codons
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG
Phenylalanine Phe F UUC UUU
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gln Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Trp W UGG
Tyrosine Tyr Y UAC UAU
In making such changes, the hydropathic index of amino acids may be
considered. The importance of the hydropathic amino acid index in conferring
interactive biologic function on a protein is generally understood in the art
(Kyte and
Doolittle, 1982, incorporated herein by reference). It is accepted that the
relative
1 o hydropathic character of the amino acid contributes to the secondary
structure of the
resultant protein, which in turn defines the interaction of the protein with
other
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molecules, for example, enzymes, substrates, receptors, DNA, antibodies,
antigens, and
the like. Each amino acid has been assigned a hydropathic index on the basis
of its
hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These
values are:
isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7);
serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate (-3.5);
glutariiine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and
arginine (-4.5).
It is known in the art that certain amino acids may be substituted by
other amino acids having a similar hydropathic index or score and still result
in a
l0 protein with similar biological activity, i. e. still obtain a biological
functionally
equivalent protein. In making such changes, the substitution of amino acids
whose
hydropathic indices are within ~2 is preferred, those within ~1 are
particularly
preferred, and those within ~0.5 are even more particularly preferred. It is
also
understood in the art that the substitution of like amino acids can be made
effectively on
the basis of hydrophilicity. U. S. Patent 4,554,101 (specifically incorporated
herein by
reference in its entirety), states that the greatest local average
hydrophilicity of a
protein, as governed by the hydrophilicity of its adjacent amino acids,
correlates with a
biological property of the protein.
As detailed in U. S. Patent 4,554,101, the following hydrophilicity
values have been assigned to amino acid residues: arginine (+3.0); lysine
(+3.0);
aspartate (+3.0 ~ 1 ); glutamate (+3.0 ~ 1 ); serine (+0.3); asparagine
(+0.2); glutamine
(+0.2); glycine (0); threonine (-0.4); proline (-0.5 ~ 1); alanine (-0.5);
histidine (-0.5);
cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine
(-1.8);
tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood
that an amino
acid can be substituted for another having a similar hydrophilicity value and
still obtain
a biologically equivalent, and in particular, an immunologically equivalent
protein. In
such changes, the substitution of amino acids whose hydrophilicity values are
within ~2
is preferred, those within ~1 are particularly preferred, and those within
~0.5 are even
more particularly preferred.
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As outlined above, amino acid substitutions are generally therefore based
on the relative similarity of the amino acid side-chain substituents, for
example, their
hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary
substitutions that
take various of the foregoing characteristics into consideration are well
.known to those
of skill in the art and include: arginine and lysine; glutamate and aspartate;
serine and
threonine; glutamine and asparagine; and valine, leucine and isoleucine.
In addition, any polynucleotide may be further modified to increase
stability in vivo. Possible modifications include, but are not limited to, the
addition of
flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2'
O-methyl
1 o rather than phosphodiesterase linkages in the backbone; and/or the
inclusion of
nontraditional bases such as inosine, queosine and wybutosine, as well as
acetyl-
methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine
and
uridine.
Amino acid substitutions may further be made on the basis of similarity
in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the
amphipathic
nature of the residues. For example, negatively charged amino acids include
aspartic
acid and glutamic acid; positively charged amino acids include lysine and
arginine; and
amino acids with uncharged polar head groups having similar hydrophilicity
values
include leucine, isoleucine and valine; glycine and alanine; asparagine and
glutamine;
2o and serine, threonine, phenylalanine and tyrosine. Other groups of amino
acids that may
represent conservative changes include: ( 1 ) ala, pro, gly, glu, asp, gln,
asn, ser, thr;
(2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his;
and (5) phe, tyr, trp,
his. A variant may also, or alternatively, contain nonconservative changes. In
a
preferred embodiment, variant polypeptides differ from a native sequence by
substitution, deletion or addition of five amino acids or fewer. Variants may
also (or
alternatively) be modified by, for example, the deletion or addition of amino
acids that
have minimal influence on the immunogenicity, secondary structure and
hydropathic
nature of the polypeptide.
As noted above, polypeptides may comprise a signal (or leader)
3o sequence at the N-terminal end of the protein, which co-translationally or
post-
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translationally directs transfer of the protein. The polypeptide may also be
conjugated
to a linker or other sequence for ease of synthesis, purification or
identification of the
polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a
solid support.
For example, a polypeptide may be conjugated to an immunoglobulin Fc region.
When comparing polypeptide sequences, two sequences are said to be
"identical" if the sequence of amino acids in the two sequences is the same
when
aligned for maximum correspondence, as described below. Comparisons between
two
sequences are typically performed by comparing the sequences over a comparison
window to identify and compare local regions of sequence similarity. A
"comparison
I o window" as used herein, refers to a segment of at least about 20
contiguous positions,
usually 30 to about 75, 40 to about 50, in which a sequence may be compared to
a
reference sequence of the same number of contiguous positions after the two
sequences
are optimally aligned.
Optimal alignment of sequences for comparison may be conducted using
I S the Megalign program in the Lasergene suite of bioinformatics software
(DNASTAR,
Inc., Madison, WI), using default parameters. This program embodies several
alignment schemes described in the following references: Dayhoff, M.O. (1978)
A
model of evolutionary change in proteins - Matrices for detecting distant
relationships.
In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National
Biomedical
2o Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J.
(1990)
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology
vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M.
(1989)
CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson,
E.D. (1971) Comb. Theor 11:105; Santou; N. Nes, M. (1987) Mol. Biol. Evol.
4:406-
25 425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the
Principles and
Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J.
and
Lipman, D.J. (1983) Proc. Natl. Acad , Sci. USA 80:726-730.
Alternatively, optimal alignment of sequences for comparison may be
conducted by the local identity algorithm of Smith and Waterman (1981) Add.
APL.
3o Math 2:482, by the identity alignment algorithm of Needleman and Wunsch
(1970) J.
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Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman
(1988)
Proc. Natl. Acad Sci. USA 85: 2444, by computerized implementations of these
algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison,
WI),
or by inspection.
One preferred example of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and BLAST 2.0
algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res.
25:3389-3402
and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and
BLAST
2.0 can be used, for example with the parameters described herein, to
determine percent
sequence identity for the polynucleotides and polypeptides of the invention.
Software
for performing BLAST analyses is publicly available through the National
Center for
Biotechnology Information. For amino acid sequences, a scoring matrix can be
used to
calculate the cumulative score. Extension of the word hits in each direction
are halted
1 s when: the cumulative alignment score falls off by the quantity X from its
maximum
achieved value; the cumulative score goes to zero or below, due to the
accumulation of
one or more negative-scoring residue alignments; or the end of either sequence
is
reached. The BLAST algorithm parameters W, T and X determine the sensitivity
and
speed of the alignment.
In one preferred approach, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a window of
comparison of at least 20 positions, wherein the portion of the polypeptide
sequence in
the comparison window may comprise additions or deletions (i.e., gaps) of 20
percent
or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the
reference
sequences (which does not comprise additions or deletions) for optimal
alignment of the
two sequences. The percentage is calculated by determining the number of
positions at
which the identical amino acid residue occurs in both sequences to yield the
number of
matched positions, dividing the number of matched positions by the total
number of
positions in the reference sequence (i.e., the window size) and multiplying
the results by
100 to yield the percentage of sequence identity.
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Within other illustrative embodiments, a polypeptide may be a fusion
polypeptide that comprises multiple polypeptides as described herein, or that
comprises
at least one polypeptide as described herein and an unrelated sequence, such
as a known
tumor protein. A fusion partner may, for example, assist in providing T helper
epitopes
(an immunological fusion partner), preferably T helper epitopes recognized by
humans,
or may assist in expressing the protein (an expression enhancer) at higher
yields than
the native recombinant protein. Certain preferred fusion partners are both
immunological and expression enhancing fusion partners. Other fusion partners
may be
selected so as to increase the solubility of the polypeptide or to enable the
polypeptide
to be targeted to desired intracellular compartments. Still further fusion
partners
include affinity tags, which facilitate purification of the polypeptide.
Fusion polypeptides may generally be prepared using standard
techniques, including chemical conjugation. Preferably, a fusion polypeptide
is
expressed as a recombinant polypeptide, allowing the production of increased
levels,
relative to a non-fused polypeptide, in an expression system. Briefly, DNA
sequences
encoding the polypeptide components may be assembled separately, and ligated
into an
appropriate expression vector. The 3' end of the DNA sequence encoding one
polypeptide component is ligated, with or without a peptide linker, to the 5'
end of a
DNA sequence encoding the second polypeptide component so that the reading
frames
of the sequences are in phase. This permits translation into a single fusion
polypeptide
that retains the biological activity of both component polypeptides.
A peptide linker sequence may be employed to separate the first and
second polypeptide components by a distance sufficient to ensure that each
polypeptide
folds into its secondary and tertiary structures. Such a peptide linker
sequence is
incorporated into the fusion polypeptide using standard techniques well known
in the
art. Suitable peptide linker sequences may be chosen based on the following
factors:
(1) their ability to adopt a flexible extended conformation; (2) their
inability to adopt a
secondary structure that could interact with functional epitopes on the first
and second
polypeptides; and (3) the lack of hydrophobic or charged residues that might
react with
3o the polypeptide functional epitopes. Preferred peptide linker sequences
contain Gly,
Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may
also be
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used in the linker sequence. Amino acid sequences which may be usefully
employed as
linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy
et al.,
Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and
U.S.
Patent No. 4,751,180. The linker sequence may generally be from 1 to about 50
amino
acids in length. Linker sequences are not required when the first and second
polypeptides have non-essential N-terminal amino acid regions that can be used
to
separate the functional domains and prevent steric interference.
The ligated DNA sequences are operably linked to suitable
transcriptional or translational regulatory elements. The regulatory elements
to responsible for expression of DNA are located only 5' to the DNA sequence
encoding
the first polypeptides. Similarly, stop codons required to end translation and
transcription termination signals are only present 3' to the DNA sequence
encoding the
second polypeptide.
The fusion polypeptide can comprise a polypeptide as described herein
together with an unrelated immunogenic protein, such as an immunogenic protein
capable of eliciting a recall response: Examples of such proteins include
tetanus,
tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl.
J. Med.,
336:86-91, 1997).
In one preferred embodiment, the immunological fusion partner is
2o derived from a Mycobacterium sp., such as a Mycobacterium tuberculosis-
derived Ral2
fragment. Ral2 compositions and methods for their use in enhancing the
expression
and/or immunogenicity of heterologous polynucleotide/polypeptide sequences is
described in U.S. Patent Application 60/158,585, the disclosure of which is
incorporated herein by reference in its entirety. Briefly, Ral2 refers to a
polynucleotide
region that is a subsequence of a Mycobacterium tuberculosis MTB32A nucleic
acid.
MTB32A is a serine protease of 32 KD molecular weight encoded by a gene in
virulent
and avirulent strains of M. tuberculosis. The nucleotide sequence and amino
acid
sequence of MTB32A have been described (for example, U.S. Patent Application
60/158,585; see also, Skeiky et al., Infection and Immun. (1999) 67:3998-4007,
3o incorporated herein by reference). C-terminal fragments of the MTB32A
coding
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sequence express at high levels and remain as a soluble polypeptides
throughout the
purification process. Moreover, Ral2 may enhance the immunogenicity of
heterologous
immunogenic polypeptides with which it is fused. One preferred Ral2 fusion
polypeptide comprises a 14 KD C-terminal fragment corresponding to amino acid
residues 192 to 323 of MTB32A. Other preferred Ral2 polynucleotides generally
comprise at least about 15 consecutive nucleotides, at least about 30
nucleotides, at least
about 60 nucleotides, at least about 100 nucleotides, at least about 200
nucleotides, or at
least about 300 nucleotides that encode a portion of a Ral2 polypeptide. Ral2
polynucleotides may comprise a native sequence (i. e., an endogenous sequence
that
to encodes a Ral2 polypeptide or a portion thereof) or may comprise a variant
of such a
sequence. Ral2 polynucleotide variants may contain one or more substitutions,
additions, deletions and/or insertions such that the biological activity of
the encoded
fusion polypeptide is not substantially diminished, relative to a fusion
polypeptide
comprising a native Ral2 polypeptide. Variants preferably exhibit at least
about 70%
identity, more preferably at least about 80% identity and most preferably at
least about
90% identity to a polynucleotide sequence that encodes a native Ral2
polypeptide or a
portion thereof.
Within other preferred embodiments, an immunological fusion partner is
derived from protein D, a surface protein of the gram-negative bacterium
Haemophilus
2o influenza B (WO 91/18926). Preferably, a protein D derivative comprises
approximately the first third of the protein (e.g., the first N-terminal 100-
110 amino
acids), and a protein D derivative may be lipidated. Within certain preferred
embodiments, the first 109 residues of a Lipoprotein D fusion partner is
included on the
N-terminus to provide the polypeptide with additional exogenous T-cell
epitopes and to
increase the expression level in E coli (thus functioning as an expression
enhancer).
The lipid tail ensures optimal presentation of the antigen to antigen
presenting cells.
Other fusion partners include the non-structural protein from influenzae
virus, NS 1
(hemaglutinin). Typically, the N-terminal 81 amino acids are used, although
different
fragments that include T-helper epitopes may be used.
3o In another embodiment, the immunological fusion partner is the protein
known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is
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derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine
amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292,
1986). LYTA is an autolysin that specifically degrades certain bonds in the
peptidoglycan backbone. The C-terminal domain of the LYTA protein is
responsible
for the affinity to the choline or to some choline analogues such as DEAE.
This
property has been exploited for the development of E. coli C-LYTA expressing
plasmids useful for expression' of fusion proteins. Purification of hybrid
proteins
containing the C-LYTA fragment at the amino terminus has been described (see
Biotechnology 10:795-798, 1992). Within a preferred embodiment, a repeat
portion of
LYTA may be incorporated into a fusion polypeptide. A repeat portion is found
in the
C-terminal region starting at residue 178. A particularly preferred repeat
portion
incorporates residues 188-305.
Yet another illustrative embodiment involves fusion polypeptides, and
the polynucleotides encoding them, wherein the fusion partner comprises a
targeting
signal capable of directing a polypeptide to the endosomal/lysosomal
compartment, as
described in U.S. Patent No. 5,633,234. An immunogenic polypeptide of the
invention,
when fused with this targeting signal, will associate more efficiently with
MHC class II
molecules and thereby provide enhanced in vivo stimulation of CD4+ T-cells
specific
for the polypeptide.
Polypeptides of the invention are prepared using any of a variety of well
known synthetic and/or recombinant techniques, the latter of which are further
described below. Polypeptides, portions and other variants generally less than
about
150 amino acids can be generated by synthetic means, using techniques well
known to
those of ordinary skill in the art. In one illustrative example, such
polypeptides are
synthesized using any of the commercially available solid-phase techniques,
such as the
Merrifield solid-phase synthesis method, where amino acids are sequentially
added to a
growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146,
1963.
Equipment for automated synthesis of polypeptides is commercially available
from
suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA),
and
may be operated according to the manufacturer's instructions.
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In general, polypeptide compositions (including fusion polypeptides) of
the invention are isolated. An "isolated" polypeptide is one that is removed
from its
original environment. For example, a naturally-occurring protein or
polypeptide is
isolated if it is separated from some or all of the coexisting materials in
the natural
system. Preferably, such polypeptides are also purified, e.g., are at least
about 90%
pure, more preferably at least about 95% pure and most preferably at least
about 99%
pure.
POLYNUCLEOTIDE COMPOSITIONS
to The present invention, in other aspects, provides polynucleotide
compositions. The terms "DNA" and "polynucleotide" are used essentially
interchangeably herein to refer to a DNA molecule that has been isolated free
of total
genomic DNA of a particular species. "Isolated," as used herein, means that a
polynucleotide is substantially away from other coding sequences, and that the
DNA
molecule does not contain large portions of unrelated coding DNA, such as
large
chromosomal fragments or other functional genes or polypeptide coding regions.
Of
course, this refers to the DNA molecule as originally isolated, and does not
exclude
genes or coding regions later added to the segment by the hand of man.
As will be understood by those skilled in the art, the polynucleotide
2o compositions of this invention can include genomic sequences, extra-genomic
and
plasmid-encoded sequences and smaller engineered gene segments that express,
or may
be adapted to express, proteins, polypeptides, peptides and the like. Such
segments may
be naturally isolated, or modified synthetically by the hand of man.
As will be also recognized by the skilled artisan, polynucleotides of the
invention may be single-stranded (coding or antisense) or double-stranded, and
may be
DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules may include
HnRNA molecules, which contain introns and correspond to a DNA molecule in a
one-
to-one manner, and mRNA molecules, which do not contain introns. Additional
coding
or non-coding sequences may, but need not, be present within a polynucleotide
of the
3o present invention, and a polynucleotide may, but need not, be linked to
other molecules
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and/or support materials.
Polynucleotides may comprise a native sequence (i.e., an endogenous
sequence that encodes a polypeptide/protein of the invention or a portion
thereof) or
may comprise a sequence that encodes a variant or derivative, preferably and
immunogenic variant or derivative, of such a sequence.
Therefore, according to another aspect of the present invention,
polynucleotide compositions are provided that comprise some or all of a
polynucleotide
sequence set forth in any one of SEQ ID NOs: 1-185 and 187-196, complements of
a
polynucleotide sequence set forth in any one of SEQ ID NOs: 1-185 and 187-196,
and
degenerate variants of a polynucleotide sequence set forth in any one of SEQ
ID NOs:
1-185 and 187-196. In certain preferred embodiments, the polynucleotide
sequences set
forth herein encode immunogenic polypeptides, as described above.
In other related embodiments, the present invention provides
polynucleotide variants having substantial identity to the sequences disclosed
herein in
SEQ ID NOs: 1-185 and 187-196, for example those comprising at least 70%
sequence
identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
or
higher, sequence identity compared to a polynucleotide sequence of this
invention using
the methods described herein, (e.g., BLAST analysis using standard parameters,
as
described below). One skilled in this art will recognize that these values can
be
2o appropriately adjusted to determine corresponding identity of proteins
encoded by two
nucleotide sequences by taking into account codon degeneracy, amino acid
similarity,
reading frame positioning and the like.
Typically, polynucleotide variants will contain one or more substitutions,
additions, deletions and/or insertions, preferably such that the
immunogenicity of the
polypeptide encoded by the variant polynucleotide is not substantially
diminished
relative to a polypeptide encoded by a polynucleotide sequence specifically
set forth
herein). The term "variants" should also be understood to encompasses
homologous
genes of xenogenic origin.
In additional embodiments, the present invention provides
3o polynucleotide fragments comprising various lengths of contiguous stretches
of
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sequence identical to or complementary to one or more of the sequences
disclosed
herein. For example, polynucleotides are provided by this invention that
comprise at
least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, S00 or 1000
or more
contiguous nucleotides of one or more of the sequences disclosed herein as
well as all
intermediate lengths there between. It will be readily understood that
"intermediate
lengths", in this context, means any length between the quoted values, such as
16, 17,
18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50,51, 52, 53, etc.; 100,
101, 102, 103,
etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-
1,000, and the
like.
l0 In another embodiment of the invention, polynucleotide compositions
are provided that are capable of hybridizing under moderate to high stringency
conditions to a polynucleotide sequence provided herein, or a fragment
thereof, or a
complementary sequence thereof. Hybridization techniques are well known in the
art of
molecular biology. For purposes of illustration, suitable moderately stringent
conditions for testing the hybridization of a polynucleotide of this invention
with other
polynucleotides include prewashing in a solution of S X SSC, 0.5% SDS, 1.0 mM
EDTA (pH 8.0); hybridizing at 50°C-60°C, 5 X SSC, overnight;
followed by washing
twice at 65°C for 20 minutes with each of 2X, O.SX and 0.2X SSC
containing 0.1%
SDS. One skilled in the art will understand that the stringency of
hybridization can be
readily manipulated, such as by altering the salt content of the hybridization
solution
and/or the temperature at which the hybridization is performed. For example,
in
another embodiment, suitable highly stringent hybridization conditions include
those
described above, with the exception that the temperature of hybridization is
increased,
e.g., to 60-65°C or 65-70°C.
In certain preferred embodiments, the polynucleotides described above,
e.g., polynucleotide variants, fragments and hybridizing sequences, encode
polypeptides
that are immunologically cross-reactive with a polypeptide sequence
specifically set
forth herein. In other preferred embodiments, such polynucleotides encode
polypeptides that have a level of immunogenic activity of at least about 50%,
preferably
at least about 70%, and more preferably at least about 90% of that for a
polypeptide
sequence specifically set forth herein.
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The polynucleotides of the present invention, or fragments thereof,
regardless of the length of the coding sequence itself, may be combined with
other
DNA sequences, such as promoters, polyadenylation signals, additional
restriction
enzyme sites, multiple cloning sites, other coding segments, and the like,
such that their
overall length may vary considerably. It is therefore contemplated that a
nucleic acid
fragment of almost any length may be employed, with the total length
preferably being
limited by the ease of preparation and use in the intended recombinant DNA
protocol.
For example, illustrative polynucleotide segments with total lengths of about
10,000,
about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about
100,
1o about 50 base pairs in length, and the like, (including all intermediate
lengths) are
contemplated to be useful in many implementations of this invention.
When comparing polynucleotide sequences, two sequences are said to be
"identical" if the sequence of nucleotides in the two sequences is the same
when aligned
for maximum correspondence, as described below. Comparisons between two
sequences are typically performed by comparing the sequences over a comparison
window to identify and compare local regions of sequence similarity. A
"comparison
window" as used herein, refers to a segment of at least about 20 contiguous
positions,
usually 30 to about 75, 40 to about 50, in which a sequence may be compared to
a
reference sequence of the same number of contiguous positions after the two
sequences
2o are optimally aligned.
Optimal alignment of sequences for comparison may be conducted using
the Megalign program in the Lasergene suite of bioinformatics software
(DNASTAR,
Inc., Madison, WI), using default parameters. This program embodies several
alignment schemes described in the following references: Dayhoff, M.O. (1978)
A
model of evolutionary change in proteins - Matrices for detecting distant
relationships.
In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National
Biomedical
Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J.
(1990)
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology
vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M.
(1989)
3o CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17;
Robinson,
E.D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol.
4:406-
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425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Principles
and
Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J.
and
Lipman, D.J. (1983) Proc. Natl. Acad , Sci. USA 80:726-730.
Alternatively, optimal alignment of sequences for comparison may be
s conducted by the local identity algorithm of Smith and Waterman (1981) Add.
APL.
Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970)
J.
Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman
(1988)
Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these
algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics
1o Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison,
WI),
or by inspection.
One preferred example of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and BLAST 2.0
algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res.
25:3389-3402
15 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST
and BLAST
2.0 can be used, for example with the parameters described herein, to
determine percent
sequence identity for the polynucleotides of the invention. Software for
performing
BLAST analyses is publicly available through the National Center for
Biotechnology
Information. In one illustrative example, cumulative scores can be calculated
using, for
20 nucleotide sequences, the parameters M (reward score for a pair of matching
residues;
always >0) and N (penalty score for mismatching residues; always <0).
Extension of
the word hits in each direction are halted when: the cumulative alignment
score falls off
by the quantity X from its maximum achieved value; the cumulative score goes
to zero
or below, due to the accumulation of one or more negative-scoring residue
alignments;
25 or the end of either sequence is reached. The BLAST algorithm parameters W,
T and X
determine the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation
(E) of
10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc.
Natl.
Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=-
4 and
3o a comparison of both strands.
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Preferably, the "percentage of sequence identity" is determined by comparing
two
optimally aligned sequences over a window of comparison of at least 20
positions,
wherein the portion of the polynucleotide sequence in the comparison window
may
comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5
to 15 percent,
or 10 to 12 percent, as compared to the reference sequences (which does not
comprise
additions or deletions) for optimal alignment of the two sequences. The
percentage is
calculated by determining the number of positions at which the identical
nucleic acid
bases occurs in both sequences to yield the number of matched positions,
dividing the
number of matched positions by the total number of positions in the reference
sequence
to (i.e., the window size) and multiplying the results by 100 to yield the
percentage of
sequence identity.
It will be appreciated by those of ordinary skill in the art that, as a result
of the degeneracy of the genetic code, there are many nucleotide sequences
that encode
a polypeptide as described herein. Some of these polynucleotides bear minimal
homology to the nucleotide sequence of any native gene. Nonetheless,
polynucleotides
that vary due to differences in codon usage are specifically contemplated by
the present
invention. Further, alleles of the genes comprising the polynucleotide
sequences
provided herein are within the scope of the present invention. Alleles are
endogenous
genes that are altered as a result of one or more mutations, such as
deletions, additions
2o and/or substitutions of nucleotides. The resulting mRNA and protein may,
but need
not, have an altered structure or function. Alleles may be identified using
standard
techniques (such as hybridization, amplification and/or database sequence
comparison).
Therefore, in another embodiment of the invention, a mutagenesis
approach, such as site-specific mutagenesis, is employed for the preparation
of
immunogenic variants and/or derivatives of the polypeptides described herein.
By this
approach, specific modifications in a polypeptide sequence can be made through
mutagenesis of the underlying polynucleotides that encode them. These
techniques
provides a straightforward approach to prepare and test sequence variants, for
example,
incorporating one or more of the foregoing considerations, by introducing one
or more
nucleotide sequence changes into the polynucleotide.
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Site-specific mutagenesis allows the production of mutants through the
use of specific oligonucleotide sequences which encode the DNA sequence of the
desired mutation, as well as a sufficient number of adjacent nucleotides, to
provide a
primer sequence of sufficient size and sequence complexity to form a stable
duplex on
both sides of the deletion junction being traversed. Mutations may be employed
in a
selected polynucleotide sequence to improve, alter, decrease, modify, or
otherwise
change the properties of the polynucleotide itself, and/or alter the
properties, activity,
composition, stability, or primary sequence of the encoded polypeptide.
In certain embodiments of the present invention, the inventors
l0 contemplate the mutagenesis of the disclosed polynucleotide sequences to
alter one or
more properties of the encoded polypeptide, such as the immunogenicity of a
polypeptide vaccine. The techniques of site-specific mutagenesis are well-
known in the
art, and are widely used to create variants of both polypeptides and
polynucleotides.
For example, site-specific mutagenesis is often used to alter a specific
portion of a DNA
molecule. In such embodiments, a primer comprising typically about 14 to about
25
nucleotides or so in length is employed, with about 5 to about 10 residues on
both sides
of the junction of the sequence being altered.
As will be appreciated by those of skill in the art, site-specific
mutagenesis techniques have often employed a phage vector that exists in both
a single
stranded and double stranded form. Typical vectors useful in site-directed
mutagenesis
include vectors such as the M 13 phage. These phage are readily
commercially-available and their use is generally well-known to those skilled
in the art.
Double-stranded plasmids are also routinely employed in site directed
mutagenesis that
eliminates the step of transferring the gene of interest from a plasmid to a
phage.
In general, site-directed mutagenesis in accordance herewith is
performed by first obtaining a single-stranded vector or melting apart of two
strands of
a double-stranded vector that includes within its sequence a DNA sequence that
encodes
the desired peptide. An oligonucleotide primer bearing the desired mutated
sequence is
prepared, generally synthetically. This primer is then annealed with the
single-stranded
3o vector, and subjected to DNA polymerizing enzymes such as E. coli
polymerase I
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Klenow fragment, in order to complete the synthesis of the mutation-bearing
strand.
Thus, a heteroduplex is formed wherein one strand encodes the original non-
mutated
sequence and the second strand bears the desired mutation. This heteroduplex
vector is
then used to transform appropriate cells, such as E coli cells, and clones are
selected
which include recombinant vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of the selected peptide-encoding
DNA segments using site-directed mutagenesis provides a means of producing
potentially useful species and is not meant to be limiting as there are other
ways in
which sequence variants. of peptides and the DNA sequences encoding them may
be
obtained. For example, recombinant vectors encoding the desired peptide
sequence
may be treated with mutagenic agents, such as hydroxylamine, to obtain
sequence
variants. Specific details regarding these methods and protocols are found in
the
teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby,
1994;
and Maniatis et al., 1982, each incorporated herein by reference, for that
purpose.
As used herein, the term "oligonucleotide directed mutagenesis
procedure" refers to template-dependent processes and vector-mediated
propagation
which result in an increase in the concentration of a specific nucleic acid
molecule
relative to its initial concentration, or in an increase in the concentration
of a detectable
signal, such as amplification. As used herein, the term "oligonucleotide
directed
2o mutagenesis procedure" is intended to refer to a process that involves the
template-dependent extension of a primer molecule. The term template dependent
process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein
the
sequence of the newly synthesized strand of nucleic acid is dictated by the
well-known
rules of complementary base pairing (see, for example, Watson, 1987).
Typically,
vector mediated methodologies involve the introduction of the nucleic acid
fragment
into a DNA or RNA vector, the clonal amplification of the vector, and the
recovery of
the amplified nucleic acid fragment. Examples of such methodologies are
provided by
U. S. Patent No. 4,237,224, specifically incorporated herein by reference in
its entirety.
In another approach for the production of polypeptide variants of the
present invention, recursive sequence recombination, as described in U.S.
Patent No.
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5,837,458, may be employed. In this approach, iterative cycles of
recombination and
screening or selection are performed to "evolve" individual polynucleotide
variants of
the invention having, for example, enhanced immunogenic activity.
In other embodiments of the present invention, the polynucleotide
sequences provided herein can be advantageously used as probes or primers for
nucleic
acid hybridization. As such, it is contemplated that nucleic acid segments
that comprise
a sequence region of at least about 15 nucleotide long contiguous sequence
that has the
same sequence as, or is complementary to, a 15 nucleotide long contiguous
sequence
disclosed herein will find particular utility. Longer contiguous identical or
1o complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200,
500, 1000
(including all intermediate lengths) and even up to full length sequences will
also be of
use in certain embodiments.
The ability of such nucleic acid probes to specifically hybridize to a
sequence of interest will enable them to be of use in detecting the presence
of
complementary sequences in a given sample. However, other uses are also
envisioned,
such as the use of the sequence information for the preparation of mutant
species
primers, or primers for use in preparing other genetic constructions.
Polynucleotide molecules having sequence regions consisting of
contiguous nucleotide stretches of 10-14, 15-20, 30, S0, or even of 100-200
nucleotides
or so (including intermediate lengths as well), identical or complementary to
a
polynucleotide sequence disclosed herein, are particularly contemplated as
hybridization probes for use in, e.g., Southern and Northern blotting. This
would allow
a gene product, or fragment thereof, to be analyzed, both in diverse cell
types and also
in various bacterial cells. The total size of fragment, as well as the size of
the
complementary stretch(es), will ultimately depend on the intended use or
application of
the particular nucleic acid segment. Smaller fragments will generally find use
in
hybridization embodiments, wherein the length of the contiguous complementary
region may be varied, such as between about 15 and about 100 nucleotides, but
larger
contiguous complementarity stretches may be used, according to the length
3o complementary sequences one wishes to detect.
27
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The use of a hybridization probe of about 15-25 nucleotides in length
allows the formation of a duplex molecule that is both stable and selective.
Molecules
having contiguous complementary sequences over stretches greater than 15 bases
in
length are generally preferred, though, in order to increase stability and
selectivity of the
s hybrid, and thereby improve the quality and degree of specific hybrid
molecules
obtained. One will generally prefer to design nucleic acid molecules having
gene-
complementary stretches of 1 S to 25 contiguous nucleotides, or even longer
where
desired.
Hybridization probes may be selected from any portion of any of the
l0 sequences disclosed herein. All that is required is to review the sequences
set forth
herein, or to any continuous portion of the sequences, from about 15-25
nucleotides in
length up to and including the full length sequence, that one wishes to
utilize as a probe
or primer. The choice of probe and primer sequences may be governed by various
factors. For example, one may wish to employ primers from towards the termini
of the
15 total sequence.
Small polynucleotide segments or fragments may be readily prepared by,
for example, directly synthesizing the fragment by chemical means, as is
commonly
practiced using an automated oligonucleotide synthesizer. Also, fragments may
be
obtained by application of nucleic acid reproduction technology, such as the
PCRTM
2o technology of U. S. Patent 4,683,202 (incorporated herein by reference), by
introducing
selected sequences into recombinant vectors for recombinant production, and by
other
recombinant DNA techniques generally known to those of skill in the art of
molecular
biology.
The nucleotide sequences of the invention may be used for their ability to
25 selectively form duplex molecules with complementary stretches of the
entire gene or
gene fragments of interest. Depending on the application envisioned, one will
typically
desire to employ varying conditions of hybridization to achieve varying
degrees of
selectivity of probe towards target sequence. For applications requiring high
selectivity,
one will typically desire to employ relatively stringent conditions to form
the hybrids,
3o e.g., one will select relatively low salt and/or high temperature
conditions, such as
28
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provided by a salt concentration of from about 0.02 M to about 0.15 M salt at
temperatures of from about 50°C to about 70°C. Such selective
conditions tolerate
little, if any, mismatch between the probe and the template or target strand,
and would
be particularly suitable for isolating related sequences.
Of course, for some applications, for example, where one desires to
prepare mutants employing a mutant primer strand hybridized to an underlying
template, less stringent (reduced stringency) hybridization conditions will
typically be
needed in order to allow formation of the heteroduplex. In these
circumstances, one
may desire to employ salt conditions such as those of from about 0.15 M to
about 0.9 M
l0 salt, at temperatures ranging from about 20°C to about 55°C.
Cross-hybridizing species
can thereby be readily identified as positively hybridizing signals with
respect to control
hybridizations. In any case, it is generally appreciated that conditions can
be rendered
more stringent by the addition of increasing amounts of formamide, which
serves to
destabilize the hybrid duplex in the same manner as increased temperature.
Thus,
hybridization conditions can be readily manipulated, and thus will generally
be a
method of choice depending on the desired results.
According to another embodiment of the present invention,
polynucleotide compositions comprising antisense oligonucleotides are
provided.
Antisense oligonucleotides have been demonstrated to be effective and targeted
inhibitors of protein synthesis, and, consequently, provide a therapeutic
approach by
which a disease can be treated by inhibiting the synthesis of proteins that
contribute to
the disease. The efficacy of antisense oligonucleotides for inhibiting protein
synthesis
is well established. For example, the synthesis of polygalactauronase and the
muscarine
type 2 acetylcholine receptor are inhibited by antisense oligonucleotides
directed to
their respective mRNA sequences (U. S. Patent 5,739,119 and U. S. Patent
5,759,829).
Further, examples of antisense inhibition have been demonstrated with the
nuclear
protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin,
STK-1,
striatal GABAA receptor and human EGF (Jaskulski et al., Science. 1988 Jun
10;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-
32; Peris et al., Brain Res Mol Brain Res. 1998 Jun 15;57(2):310-20; U. S.
Patent
5,801,154; U.S. Patent 5,789,573; U. S. Patent 5,718,709 and U.S. Patent
5,610,288).
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Antisense constructs have also been described that inhibit and can be used to
treat a
variety of abnormal cellular proliferations, e.g. cancer (U. S. Patent
5,747,470; U. S.
Patent 5,591,317 and U. S. Patent 5,783,683).
Therefore, in certain embodiments, the present invention provides
oligonucleotide sequences that comprise all, or a portion of, any sequence
that is
capable of specifically binding to polynucleotide sequence described herein,
or a
complement thereof. In one embodiment, the antisense oligonucleotides comprise
DNA
or derivatives thereof. In another embodiment, the oligonucleotides comprise
RNA or
derivatives thereof. In a third embodiment, the oligonucleotides are modified
DNAs
l0 comprising a phosphorothioated modified backbone. In a fourth embodiment,
the
oligonucleotide sequences comprise peptide nucleic acids or derivatives
thereof. In
each case, preferred compositions. comprise a sequence region that is
complementary,
and more preferably substantially-complementary, and even more preferably,
completely complementary to one or more portions of polynucleotides disclosed
herein.
Selection of antisense compositions specific for a given gene sequence is
based upon analysis of the chosen target sequence (i. e. in these illustrative
examples the
rat and human sequences) and determination of secondary structure, Tm, binding
energy,
relative stability, and antisense compositions were selected based upon their
relative
inability to form dimers, hairpins, or other secondary structures that would
reduce or
2o prohibit specific binding to the target mRNA in a host cell.
Highly preferred target regions of the mRNA, are those which are at or
near the AUG translation initiation codon, and those sequences which are
substantially
complementary to 5' regions of the mRNA. These secondary structure analyses
and
target site selection considerations can be performed, for example, using v.4
of the
OLIGO primer analysis software and/or the BLASTN 2Ø5 algorithm software
(Altschul et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-402).
The use of an antisense delivery method employing a short peptide
vector, termed MPG (27 residues), is also contemplated. The MPG peptide
contains a
hydrophobic domain derived from the fusion sequence of HIV gp41 and a
hydrophilic
3o domain from the nuclear localization sequence of SV40 T-antigen (Morris et
al.,
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
Nucleic Acids Res. 1997 Jul 15;25(14):2730-6). It has been demonstrated that
several
molecules of the MPG peptide coat the antisense oligonucleotides and can be
delivered
into cultured mammalian cells in less than 1 hour with relatively high
efficiency (90%).
Further, the interaction with MPG strongly increases both the stability of the
oligonucleotide to nuclease and the ability to cross the plasma membrane.
According to another embodiment of the invention, the polynucleotide
compositions described herein are used in the design and preparation of
ribozyme
molecules for inhibiting expression of the tumor polypeptides and proteins of
the
present invention in tumor cells. Ribozymes are RNA-protein complexes that
cleave
to nucleic acids in a site-specific fashion. Ribozymes have specific catalytic
domains that
possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci U S A. 1987
Dec;84(24):8788-92; Forster and Symons, Cell. 1987 Apr 24;49(2):211-20). For
example, a large number of ribozymes accelerate phosphoester transfer
reactions with a
high degree of specificity, often cleaving only one of several phosphoesters
in an
oligonucleotide substrate (Cech et al., Cell. 1981 Dec;27(3 Pt 2):487-96;
Michel and
Westhof, J Mol Biol. 1990 Dec 5;216(3):585-610; Reinhold-Hurek and Shub,
Nature.
1992 May 14;357(6374):173-6). This specificity has been attributed to the
requirement
that the substrate bind via specific base-pairing interactions to the internal
guide
sequence ("IGS") of the ribozyme prior to chemical reaction.
2o Six basic varieties of naturally-occurring enzymatic RNAs are known
presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in
traps (and
thus can cleave other RNA molecules) under physiological conditions. In
general,
enzymatic nucleic acids act by first binding to a target RNA. Such binding
occurs
through the target binding portion of a enzymatic nucleic acid which is held
in close
proximity to an enzymatic portion of the molecule that acts to cleave the
target RNA.
Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA
through
complementary base-pairing, and once bound to the correct site, acts
enzymatically to
cut the target RNA. Strategic cleavage of such a target RNA will destroy its
ability to
direct synthesis of an encoded protein. After an enzymatic nucleic acid has
bound and
3o cleaved its RNA target, it is released from that RNA to search for another
target and can
repeatedly bind and cleave new targets.
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The enzymatic nature of a ribozyme is advantageous over many
technologies, such as antisense technology (where a nucleic acid molecule
simply binds
to a nucleic acid target to block its translation) since the concentration of
ribozyme
necessary to affect a therapeutic treatment is lower than that of an antisense
oligonucleotide. This advantage reflects the ability of the ribozyme to act
enzymatically. Thus, a single ribozyme molecule is able to cleave many
molecules of
target RNA. In addition, the ribozyme is a highly specific inhibitor, with the
specificity
of inhibition depending not only on the base pairing mechanism of binding to
the target
RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or
base-
1o substitutions, near the site of cleavage can completely eliminate catalytic
activity of a
ribozyme. Similar mismatches in antisense molecules do not prevent their
action
(Woolf et al., Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7305-9). Thus, the
specificity of action of a ribozyme is greater than that of an antisense
oligonucleotide
binding the same RNA site.
~ 5 The enzymatic nucleic acid molecule may be formed in a hammerhead,
hairpin, a hepatitis S virus, group I intron or RNaseP RNA (in association
with an RNA
guide sequence) or Neurospora VS RNA motif. Examples of hammerhead motifs are
described by Rossi et al. Nucleic Acids Res. 1992 Sep 11;20(17):4559-65.
Examples of
hairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP
0360257),
2o Hampel and Tritz, Biochemistry 1989 Jun 13;28(12):4929-33; Hampel et al.,
Nucleic
Acids Res. 1990 Jan 25;18(2):299-304 and U. S. Patent 5,631,359. An example of
the
hepatitis b virus motif is described by Perrotta and Been, Biochemistry. 1992
Dec
1;31 (47):11843-52; an example of the RNaseP motif is described by Guerrier-
Takada
et al., Cell. 1983 Dec;35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motif is
25 described by Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96;
Saville and
Collins, Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8826-30; Collins and
Olive,
Biochemistry. 1993 Mar 23;32(11):2795-9); and an example of the Group I intron
is
described in (U. S. Patent 4,987,071). All that is important in an enzymatic
nucleic acid
molecule of this invention is that it has a specific substrate binding site
which is
30 complementary to one or more of the target gene RNA regions, and that it
have
nucleotide sequences within or surrounding that substrate binding site which
impart an
32
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RNA cleaving activity to the molecule. Thus the ribozyme constructs need not
be
limited to specific motifs mentioned herein.
Ribozymes may be designed as described in Int. Pat. Appl. Publ. No.
WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specifically
incorporated herein by reference) and synthesized to be tested in vitro and in
vivo, as
described. Such ribozymes can also be optimized for delivery. While specific
examples are provided, those in the art will recognize that equivalent RNA
targets in
other species can be utilized when necessary.
Ribozyme activity can be optimized by altering the length of the
1 o ribozyme binding arms, or chemically synthesizing ribozymes with
modifications that
prevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl.
Publ. No. WO
92/07065; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO
91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U. S. Patent 5,334,711; and
Int. Pat.
Appl. Publ. No. WO 94/13688, which describe various chemical modifications
that can
~ 5 be made to the sugar moieties of enzymatic RNA molecules), modifications
which
enhance their efficacy in cells, and removal of stem II bases to shorten RNA
synthesis
times and reduce chemical requirements.
Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes the
general methods for delivery of enzymatic RNA molecules. Ribozymes may be
20 administered to cells by a variety of methods known to those familiar to
the art,
including, but not restricted to, encapsulation in liposomes, by
iontophoresis, or by
incorporation into other vehicles, such as hydrogels, cyclodextrins,
biodegradable
nanocapsules, and bioadhesive microspheres. For some indications, ribozymes
may be
directly delivered ex vivo to cells or tissues with or without the
aforementioned vehicles.
25 Alternatively, the RNA/vehicle combination may be locally delivered by
direct
inhalation, by direct injection or by use of a catheter, infusion pump or
stmt. Other
routes of delivery include, but are not limited to, intravascular,
intramuscular,
subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill
form), topical,
systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed
descriptions
30 of ribozyme delivery and administration are provided in Int. Pat. Appl.
Publ. No. WO
33
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WO 01/18046 PCT/US00/24827
94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically
incorporated
herein by reference.
Another means of accumulating high concentrations of a ribozyme(s)
within cells is to incorporate the ribozyme-encoding sequences into a DNA
expression
vector. Transcription of the ribozyme sequences are driven from a promoter for
eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA
polymerase
III (pol III). Transcripts from pol II or pol III promoters will be expressed
at high levels
in all cells; the levels of a given pol II promoter in a given cell type will
depend on the
nature of the gene regulatory sequences (enhancers, silencers, etc. ) present
nearby.
Prokaryotic RNA polymerase promoters may also be used, providing that the
prokaryotic RNA polymerase enzyme is expressed in the appropriate cells .
Ribozymes
expressed from such promoters have been shown to function in mammalian cells.
Such
transcription units can be incorporated into a variety of vectors for
introduction into
mammalian cells, including but not restricted to, plasmid DNA vectors, viral
DNA
vectors (such as adenovirus or adeno-associated vectors), or viral RNA vectors
(such as
retroviral, semliki forest virus, sindbis virus vectors).
In another embodiment of the invention, peptide nucleic acids (PNAs)
compositions are provided. PNA is a DNA mimic in which the nucleobases are
attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid
Drug
2o Dev. 1997 7(4) 431-37). PNA is able to be utilized in a number methods that
traditionally have used RNA or DNA. Often PNA sequences perform better in
techniques than the corresponding RNA or DNA sequences and have utilities that
are
not inherent to RNA or DNA. A review of PNA including methods of making,
characteristics of, and methods of using, is provided by Corey (Trends
Biotechnol 1997
Jun;lS(6):224-9). As such, in certain embodiments, one may prepare PNA
sequences
that are complementary to one or more portions of the ACE mRNA sequence, and
such
PNA compositions may be used to regulate, alter, decrease, or reduce the
translation of
ACE-specific mRNA, and thereby alter the level of ACE activity in a host cell
to which
such PNA compositions have been administered.
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PNAs have 2-aminoethyl-glycine linkages replacing the normal
phosphodiester backbone of DNA (Nielsen et al., Science 1991 Dec
6;254(5037):1497-
500; Hanvey et al., Science. 1992 Nov 27;258(5087):1481-5; Hyrup and Nielsen,
Bioorg Med Chem. 1996 Jan;4(1):S-23). This chemistry has three important
consequences: firstly, in contrast to DNA or phosphorothioate
oligonucleotides, PNAs
are neutral molecules; secondly, PNAs are achiral, which avoids the need to
develop a
stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc or
Fmoc
protocols for solid-phase peptide synthesis, although other methods, including
a
modified Merrifield method, have been used.
to PNA monomers or ready-made oligomers are commercially available
from PerSeptive Biosystems (Framingham, MA). PNA syntheses by either Boc or
Fmoc protocols are straightforward using manual or automated protocols (Norton
et al.,
Bioorg Med Chem. 1995 Apr;3(4):437-45). The manual protocol lends itself to
the
production of chemically modified PNAs or the simultaneous synthesis of
families of
closely related PNAs.
As with peptide synthesis, the success of a particular PNA synthesis will
depend on the properties of the chosen sequence. For example, while in theory
PNAs
can incorporate any combination of nucleotide bases, the presence of adjacent
purines
can lead to deletions of one or more residues in the product. In expectation
of this
2o difficulty, it is suggested that, in producing PNAs with adjacent purines,
one should
repeat the coupling of residues likely to be added inefficiently. This should
be followed
by the purification of PNAs by reverse-phase high-pressure liquid
chromatography,
providing yields and purity of product similar to those observed during the
synthesis of
peptides.
Modifications of PNAs for a given application may be accomplished by
coupling amino acids during solid-phase synthesis or by attaching compounds
that
contain a carboxylic acid group to the exposed N-terminal amine.
Alternatively, PNAs
can be modified after synthesis by coupling to an introduced lysine or
cysteine. The
ease with which PNAs can be modified facilitates optimization for better
solubility or
3o for specific ftmctional requirements. Once synthesized, the identity of
PNAs and their
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
derivatives can be confirmed by mass spectrometry. Several studies have made
and
utilized modifications of PNAs (for example, Norton et al., Bioorg Med Chem.
1995
Apr;3(4):437-45; Petersen et al., J Pept Sci. 1995 May-Jun;l(3):175-83; Orum
et al.,
Biotechniques. 1995 Sep;l9(3):472-80; Footer et al., Biochemistry. 1996 Aug
20;35(33):10673-9; Griffith et al., Nucleic Acids Res. 1995 Aug 11;23(15):3003-
8;
Pardridge et al., Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5592-6; Boffa et
al.,
Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):1901-5; Gambacorti-Passerini et
al.,
Blood. 1996 Aug 15;88(4):1411-7; Armitage et al., Proc Natl Acad Sci U S A.
1997
Nov 11;94(23):12320-S; Seeger et al., Biotechniques. 1997 Sep;23(3):512-7).
U.5.
t o Patent No. 5,700,922 discusses PNA-DNA-PNA chimeric molecules and their
uses in
diagnostics, modulating protein in organisms, and treatment of conditions
susceptible to
therapeutics.
Methods of characterizing the antisense binding properties of PNAs are
discussed in Rose (Anal Chem. 1993 Dec 15;65(24):3545-9) and Jensen et al.
(Biochemistry. 1997 Apr 22;36(16):5072-7). Rose uses capillary gel
electrophoresis to
determine binding of PNAs to their complementary oligonucleotide, measuring
the
relative binding kinetics and stoichiometry. Similar types of measurements
were made
by Jerisen et al. using BIAcoreTM technology.
Other applications of PNAs that have been described and will be
2o apparent to the skilled artisan include use in DNA strand invasion,
antisense inhibition,
mutational analysis, enhancers of transcription, nucleic acid purification,
isolation of
transcriptionally active genes, blocking of transcription factor binding,
genome
cleavage, biosensors, in situ hybridization, and the like.
POLYNUCLEOTIDE IDENTIFICATION ~ CHARACTERIZATION AND EXPRESSION
Polynucleotides compositions of the present invention may be identified,
prepared and/or manipulated using any of a variety of well established
techniques (see
generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring
Harbor Laboratories, Cold Spring Harbor, NY, 1989, and other like references).
For
3o example, a polynucleotide may be identified, as described in more detail
below, by
36
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WO 01/18046 PCT/US00/24827
screening a microarray of cDNAs for tumor-associated expression (i.e.,
expression that
is at least two fold greater in a tumor than in normal tissue, as determined
using a
representative assay provided herein). Such screens may be performed, for
example,
using a Synteni microarray (Palo Alto, CA) according to the manufacturer's
instructions
(and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA
93:10614-
10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997).
Alternatively, polynucleotides may be amplified from cDNA prepared from cells
expressing the proteins described herein, such as tumor cells.
Many template dependent processes are available to amplify a target
sequences of interest present in a sample. One of the best known amplification
methods
is the polymerase chain reaction (PCRT~ which is described in detail in U.S.
Patent
Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein
by
reference in its entirety. Briefly, in PCRTM, two primer sequences are
prepared which
are complementary to regions on opposite complementary strands of the target
sequence. An excess of deoxynucleoside triphosphates is added to a reaction
mixture
along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is
present
in a sample, the primers will bind to the target and the polymerase will cause
the
primers to be extended along the target sequence by adding on nucleotides. By
raising
and lowering the temperature of the reaction mixture, the extended primers
will
2o dissociate from the target to form reaction products, excess primers will
bind to the
target and to the reaction product and the process is repeated. Preferably
reverse
transcription and PCRTM amplification procedure may be performed in order to
quantify
the amount of mRNA amplified. Polymerase chain reaction methodologies are well
known in the art.
z5 Any of a number of other template dependent processes, many of which
are variations of the PCR TM amplification technique, are readily known and
available in
the art. Illustratively, some such methods include the ligase chain reaction
(referred to
as LCR), described, for example, in Eur. Pat. Appl. Publ. No. 320,308 and U.S.
Patent
No. 4,883,750; Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.
3o PCT/LJS87/00880; Strand Displacement Amplification (SDA) and Repair Chain
Reaction (RCR). Still other amplification methods are described in Great
Britain Pat.
37
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WO 01/18046 PCT/US00/24827
Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025.
Other
nucleic acid amplification procedures include transcription-based
amplification systems
(TAS) (PCT Intl. Pat. Appl. Publ. No. WO 88/10315), including nucleic acid
sequence
based amplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822
describes a
nucleic acid amplification process involving cyclically synthesizing single-
stranded
RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA). PCT Intl. Pat. Appl.
Publ. No. WO 89/06700 describes a nucleic acid sequence amplification scheme
based
on the hybridization of a promoter/primer sequence to a target single-stranded
DNA
("ssDNA") followed by transcription of many RNA copies of the sequence. Other
t 0 amplification methods such as "RACE" (Frohman, 1990), and "one-sided PCR"
(Ohara,
1989) are also well-known to those of skill in the art.
An amplified portion of a polynucleotide of the present invention may be
used to isolate a full length gene from a suitable library (e.g., a tumor cDNA
library)
using well known techniques. Within such techniques, a library (cDNA or
genomic) is
~ 5 screened using one or more polynucleotide probes or primers suitable for
amplification.
Preferably, a library is size-selected to include larger molecules. Random
primed
libraries may also be preferred for identifying S' and upstream regions of
genes.
Genomic libraries are preferred for obtaining introns and extending 5'
sequences.
For hybridization techniques, a partial sequence may be labeled (e.g., by
20 nick-translation or end-labeling with'ZP) using well known techniques. A
bacterial or
bacteriophage library is then generally screened by hybridizing filters
containing
denatured bacterial colonies (or lawns containing phage plaques) with the
labeled probe
(see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor
Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques
are
25 selected and expanded, and the DNA is isolated for further analysis. cDNA
clones may
be analyzed to determine the amount of additional sequence by, for example,
PCR using
a primer from the partial sequence and a primer from the vector. Restriction
maps and
partial sequences may be generated to identify one or more overlapping clones.
The
complete sequence may then be determined using standard techniques, which may
30 involve generating a series of deletion clones. The resulting overlapping
sequences can
then assembled into a single contiguous sequence. A full length cDNA molecule
can be
38
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WO 01/18046 PCT/US00/24827
generated by ligating suitable fragments, using well known techniques.
Alternatively, amplification techniques, such as those described above,
can be useful for obtaining a full length coding sequence from a partial cDNA
sequence.
One such amplification technique is inverse PCR (see Triglia et al., Nucl.
Acids Res.
16:8186, 1988), which uses restriction enzymes to generate a fragment in the
known
region of the gene. The fragment is then circularized by intramolecular
ligation and
used as a template for PCR with divergent primers derived from the known
region.
Within an alternative approach, sequences adjacent to a partial sequence may
be
retrieved by amplification with a primer to a linker sequence and a primer
specific to a
1o known region. The amplified sequences are typically subjected to a second
round of
amplification with the same linker primer and a second primer specific to the
known
region. A variation on this procedure, which employs two primers that initiate
extension in opposite directions from the known sequence, is described in WO
96/38591. Another such technique is known as "rapid amplification of cDNA
ends" or
RACE. This technique involves the use of an internal primer and an external
primer,
which hybridizes to a polyA region or vector sequence, to identify sequences
that are 5'
and 3' of a known sequence. Additional techniques include capture PCR
(Lagerstrom et
al., PCR Methods Applic. 1:l l l-19, 1991) and walking PCR (Parker et al.,
Nucl. Acids.
Res. 19:3055-60, 1991). Other methods employing amplification may also be
employed to obtain a full length cDNA sequence.
In certain instances, it is possible to obtain a full length cDNA sequence
by analysis of sequences provided in an expressed sequence tag (EST) database,
such as
that available from GenBank. Searches for overlapping ESTs may generally be
performed using well known programs (e.g., NCBI BLAST searches), and such ESTs
may be used to generate a contiguous full length sequence. Full length DNA
sequences
may also be obtained by analysis of genomic fragments.
In other embodiments of the invention, polynucleotide sequences or
fragments thereof which encode polypeptides of the invention, or fusion
proteins or
functional equivalents thereof, may be used in recombinant DNA molecules to
direct
expression of a polypeptide in appropriate host cells. Due to the inherent
degeneracy of
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the genetic code, other DNA sequences that encode substantially the same or a
functionally equivalent amino acid sequence may be produced and these
sequences may
be used to clone and express a given polypeptide.
As will be understood by those of skill in the art, it may be advantageous
in some instances to produce polypeptide-encoding nucleotide sequences
possessing
non-naturally occurring codons. For example, codons preferred by a particular
prokaryotic or eukaryotic host can be selected to increase the rate of protein
expression
or to produce a recombinant RNA transcript having desirable properties, such
as a half
life which is longer than that of a transcript generated from the naturally
occurring
1o sequence.
Moreover, the polynucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to alter
polypeptide
encoding sequences for a variety of reasons, including but not limited to,
alterations
which modify the cloning, processing, and/or expression of the gene product.
For
example, DNA shuffling by random fragmentation and PCR reassembly of gene
fragments and synthetic oligonucleotides may be used to engineer the
nucleotide
sequences. In addition, site-directed mutagenesis may be used to insert new
restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, or
introduce mutations, and so forth.
In another embodiment of the invention, natural, modified, or
recombinant nucleic acid sequences may be ligated to a heterologous sequence
to
encode a fusion protein. For example, to screen peptide libraries for
inhibitors of
polypeptide activity, it may be useful to encode a chimeric protein that can
be
recognized by a commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the polypeptide-encoding
sequence and the heterologous protein sequence, so that the polypeptide may be
cleaved and purified away from the heterologous moiety.
Sequences encoding a desired polypeptide may be synthesized, in whole
or in part, using chemical methods well known in the art (see Caruthers, M. H.
et al.
(1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids
Res.
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Symp. Ser. 225-232). Alternatively, the protein itself may be produced using
chemical
methods to synthesize the amino acid sequence of a polypeptide, or a portion
thereof.
For example, peptide synthesis can be performed using various solid-phase
techniques
(Roberge, J. Y. et al. (1995) Science 269:202-204) and automated synthesis may
be
achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer,
Palo
Alto, CA).
A newly synthesized peptide may be substantially purified by
preparative high performance liquid chromatography (e.g., Creighton, T. (1983)
Proteins, Structures and Molecular Principles, WH Freeman and Co., New York,
N.Y.)
to or other comparable techniques available in the art. The composition of the
synthetic
peptides may be confirmed by amino acid analysis or sequencing (e.g., the
Edman
degradation procedure). Additionally, the amino acid sequence of a
polypeptide, or any
part thereof, may be altered during direct synthesis and/or combined using
chemical
methods with sequences from other proteins, or any part thereof, to produce a
variant
polypeptide.
In order to express a desired polypeptide, the nucleotide sequences
encoding the polypeptide, or functional equivalents, may be inserted into
appropriate
expression vector, i.e., a vector which contains the necessary elements for
the
transcription and translation of the inserted coding sequence. Methods which
are well
2o known to those skilled in the art may be used to construct expression
vectors containing
sequences encoding a polypeptide of interest and appropriate transcriptional
and
translational control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic recombination. Such
techniques
are described, for example, in Sambrook, J. et al. (1989) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F.
M. et
al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New
York.
N.Y.
A variety of expression vector/host systems may be utilized to contain
and express polynucleotide sequences. These include, but are not limited to,
3o microorganisms such as bacteria transformed with recombinant bacteriophage,
plasmid,
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or cosmid DNA expression vectors; yeast transformed with yeast expression
vectors;
insect cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell
systems transformed with virus expression vectors (e.g., cauliflower mosaic
virus,
CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g.,
Ti or
pBR322 plasmids); or animal cell systems.
The "control elements" or "regulatory sequences" present in an
expression vector are those non-translated regions of the vector--enhancers,
promoters,
5' and 3' untranslated regions--which interact with host cellular proteins to
carry out
transcription and translation. Such elements may vary in their strength and
specificity.
l0 Depending on the vector system and host utilized, any number of suitable
transcription
and translation elements, including constitutive and inducible promoters, may
be used.
For example, when cloning in bacterial systems, inducible promoters such as
the hybrid
lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or
PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) and the like may be used. In
mammalian cell systems, promoters from mammalian genes or from mammalian
viruses are generally preferred. If it is necessary to generate a cell line
that contains
multiple copies of the sequence encoding a polypeptide, vectors based on SV40
or EBV
may be advantageously used with an appropriate selectable marker.
In bacterial systems, any of a number of expression vectors may be
2o selected depending upon the use intended for the expressed polypeptide. For
example,
when large quantities are needed, for example for the induction of antibodies,
vectors
which direct high level expression of fusion proteins that are readily
purified may be
used. Such vectors include, but are not limited to, the multifunctional E.
coli cloning
and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence
encoding the polypeptide of interest may be ligated into the vector in frame
with
sequences for the amino-terminal Met and the subsequent 7 residues of .beta.-
galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G.
and S.
M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX Vectors
(Promega, Madison, Wis.) may also be used to express foreign polypeptides as
fusion
3o proteins with glutathione S-transferase (GST). In general, such fusion
proteins are
soluble and can easily be purified from lysed cells by adsorption to
glutathione-agarose
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beads followed by elution in the presence of free glutathione. Proteins made
in such
systems may be designed to include heparin, thrombin, or factor XA protease
cleavage
sites so that the cloned polypeptide of interest can be released from the GST
moiety at
will.
In the yeast, Saccharomyces cerevisiae, a number of vectors containing
constitutive or inducible promoters such as alpha factor, alcohol oxidase, and
PGH may
be used. For reviews, see Ausubel et al. (supra) and Grant et al. (1987)
Methods
Enzymol. 153 :516-544.
In cases where plant expression vectors are used, the expression of
sequences encoding polypeptides may be driven by any of a number of promoters.
For
example, viral promoters such as the 35S and 19S promoters of CaMV may be used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J. 6:307-311. Alternatively, plant promoters such as the small
subunit of
RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.
~s 3:1671-1680; Brogue, R. et al. (1984) Science 224:838-843; and Winter, J.
et al. (1991)
Results Probl. Cell Differ. 17:85-105). These constructs can be introduced
into plant
cells by direct DNA transformation or pathogen-mediated transfection. Such
techniques
are described in a number of generally available reviews (see, for example,
Hobbs, S. or
Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw
20 Hill, New York, N.Y.; pp. 191-185 and 187-196).
An insect system may also be used to express a polypeptide of interest.
For example, in one such system, Autographa californica nuclear polyhedrosis
virus
(AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda
cells or
in Trichoplusia larvae. The sequences encoding the polypeptide may be cloned
into a
25 non-essential region of the virus, such as the polyhedrin gene, and placed
under control
of the polyhedrin promoter. Successful insertion of the polypeptide-encoding
sequence
will render the polyhedrin gene inactive and produce recombinant virus lacking
coat
protein. The recombinant viruses may then be used to infect, for example, S.
frugiperda
cells or Trichoplusia larvae in which the polypeptide of interest may be
expressed
30 (Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. 91 :3224-3227).
43
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In mammalian host cells, a number of viral-based expression systems are
generally available. For example, in cases where an adenovirus is used as an
expression
vector, sequences encoding a polypeptide of interest may be ligated into an
adenovirus
transcription/translation complex consisting of the late promoter and
tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used
to obtain a viable virus which is capable of expressing the polypeptide in
infected host
cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In
addition,
transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be
used
to increase expression in mammalian host cells.
l0 Specific initiation signals may also be used to achieve more efficient
translation of sequences encoding a polypeptide of interest. Such signals
include the
ATG initiation codon and adjacent sequences. In cases where sequences encoding
the
polypeptide, its initiation codon, and upstream sequences are inserted into
the
appropriate expression vector, no additional transcriptional or translational
control
signals may be needed. However, in cases where only coding sequence, or a
portion
thereof, is inserted, exogenous translational control signals including the
ATG initiation
codon should be provided. Furthermore, the initiation codon should be in the
correct
reading frame to ensure translation of the entire insert. Exogenous
translational
elements and initiation codons may be of various origins, both natural and
synthetic.
The efficiency of expression may be enhanced by the inclusion of enhancers
which are
appropriate for the particular cell system which is used, such as those
described in the
literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).
In addition, a host cell strain may be chosen for its ability to modulate
the expression of the inserted sequences or to process the expressed protein
in the
desired fashion. Such modifications of the polypeptide include, but are not
limited to,
acetylation, carboxylation. glycosylation, phosphorylation, lipidation, and
acylation.
Post-translational processing which cleaves a "prepro" form of the protein may
also be
used to facilitate correct insertion, folding and/or function. Different host
cells such as
CHO, COS, HeLa, MDCK, HEK293, and WI38, which have specific cellular
3o machinery and characteristic mechanisms for such post-translational
activities, may be
chosen to ensure the correct modification and processing of the foreign
protein.
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For long-term, high-yield production of recombinant proteins, stable
expression is generally preferred. For example, cell lines which stably
express a
polynucleotide of interest may be transformed using expression vectors which
may
contain viral origins of replication and/or endogenous expression elements and
a
selectable marker gene on the same or on a separate vector. Following the
introduction
of the vector, cells may be allowed to grow for 1-2 days in an enriched media
before
they are switched to selective media. The purpose of the selectable marker is
to confer
resistance to selection, and its presence allows growth and recovery of cells
which
successfully express the introduced sequences. Resistant clones of stably
transformed
1 o cells may be proliferated using tissue culture techniques appropriate to
the cell type.
Any number of selection systems may be used to recover transformed
cell lines. These include, but are not limited to, the herpes simplex virus
thymidine
kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine
phosphoribosyltransferase
(Lowy, I. et al. (1990) Cell 22:817-23) genes which can be employed in tk<sup>-</sup>
or
aprt<sup>-</sup> cells, respectively. Also, antimetabolite, antibiotic or herbicide
resistance can
be used as the basis for selection; for example, dhfr which confers resistance
to
methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70);
npt, which
confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-
Garapin, F. et
al (1981) J. Mol. Biol. 150:1-14); and als or pat, which confer resistance to
2o chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry,
supra).
Additional selectable genes have been described, for example, trpB, which
allows cells
to utilize indole in place of tryptophan, or hisD, which allows cells to
utilize histinol in
place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad.
Sci.
85:8047-51 ). The use of visible markers has gained popularity with such
markers as
anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its
substrate
luciferin, being widely used not only to identify transformants, but also to
quantify the
amount of transient or stable protein expression attributable to a specific
vector system
(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).
Although the presence/absence of marker gene expression suggests that
3o the gene of interest is also present, its presence and expression may need
to be
confirmed. For example, if the sequence encoding a polypeptide is inserted
within a
CA 02384499 2002-03-08
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marker gene sequence, recombinant cells containing sequences can be identified
by the
absence of marker gene function. Alternatively, a marker gene can be placed in
tandem
with a polypeptide-encoding sequence under the control of a single promoter.
Expression of the marker gene in response to induction or selection usually
indicates
expression of the tandem gene as well.
Alternatively, host cells that contain and express a desired
polynucleotide sequence may be identified by a variety of procedures known to
those of
skill in the art. These procedures include, but are not limited to, DNA-DNA or
DNA-
RNA hybridizations and protein bioassay or immunoassay techniques which
include,
1o for example, membrane, solution, or chip based technologies for the
detection and/or
quantification of nucleic acid or protein.
A vaxiety of protocols for detecting and measuring the expression of
polynucleotide-encoded products, using either polyclonal or monoclonal
antibodies
specific for the product are known in the art. Examples include enzyme-linked
~ 5 immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence
activated
cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal
antibodies reactive to two non-interfering epitopes on a given polypeptide may
be
preferred for some applications, but a competitive binding assay may also be
employed.
These and other assays are described, among other places, in Hampton, R. et
al. (1990;
2o Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) and
Maddox,
D. E. et al. (1983; J. Exp. Med. 158:1211-1216).
A wide variety of labels and conjugation techniques are known by those
skilled in the art and may be used in various nucleic acid and amino acid
assays. Means
for producing labeled hybridization or PCR probes for detecting sequences
related to
25 polynucleotides include oligolabeling, nick translation, end-labeling or
PCR
amplification using a labeled nucleotide. Alternatively, the sequences, or any
portions
thereof may be cloned into a vector for the production of an mRNA probe. Such
vectors
are known in the art, are commercially available, and may be used to
synthesize RNA
probes in vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6
3o and labeled nucleotides. These procedures may be conducted using a variety
of
46
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commercially available kits. Suitable reporter molecules or labels, which may
be used
include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic
agents
as well as substrates, cofactors, inhibitors, magnetic particles, and the
like.
Host cells transformed with a polynucleotide sequence of interest may be
cultured under conditions suitable for the expression and recovery of the
protein from
cell culture. The protein produced by a recombinant cell may be secreted or
contained
intracellularly depending on the sequence and/or the vector used. As will be
understood
by those of skill in the art, expression vectors containing polynucleotides of
the
invention may be designed to contain signal sequences which direct secretion
of the
1 o encoded polypeptide through a prokaryotic or eukaryotic cell membrane.
Other
recombinant constructions may be used to join sequences encoding a polypeptide
of
interest to nucleotide sequence encoding a polypeptide domain which will
facilitate
purification of soluble proteins. Such purification facilitating domains
include, but are
not limited to, metal chelating peptides such as histidine-tryptophan modules
that allow
purification on immobilized metals, protein A domains that allow purification
on
immobilized immunoglobulin, and the domain utilized in the FLAGS
extension/affinity
purification system (Immunex Corp., Seattle, Wash.). The inclusion of
cleavable linker
sequences such as those specific for Factor XA or enterokinase (Invitrogen.
San Diego,
Calif.) between the purification domain and the encoded polypeptide may be
used to
2o facilitate purification. One such expression vector provides for expression
of a fusion
protein containing a polypeptide of interest and a nucleic acid encoding 6
histidine
residues preceding a thioredoxin or an enterokinase cleavage site. The
histidine residues
facilitate purification on IMIAC (immobilized metal ion affinity
chromatography) as
described in Porath, J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the
enterokinase
cleavage site provides a means for purifying the desired polypeptide from the
fusion
protein. A discussion of vectors which contain fusion proteins is provided in
Kroll, D. J.
et al. (1993; DNA Cell Biol. 12:441-453).
In addition to recombinant production methods, polypeptides of the
invention, and fragments thereof, may be produced by direct peptide synthesis
using
3o solid-phase techniques (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-
2154). Protein
synthesis may be performed using manual techniques or by automation. Automated
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synthesis may be achieved, for example, using Applied Biosystems 431A Peptide
Synthesizer (Perkin Elmer). Alternatively, various fragments may be chemically
synthesized separately and combined using chemical methods to produce the full
length
molecule.
ANTIBODY COMPOSITIONS, FRAGMENTS THEREOF AND OTHER BINDING AGENTS
According to another aspect, the present invention further provides
binding agents, such as antibodies and antigen-binding fragments thereof, that
exhibit
immunological binding to a tumor polypeptide disclosed herein, or to a
portion, variant
l0 or derivative thereof. An antibody, or antigen-binding fragment thereof, is
said to
"specifically bind," "immunogically bind," and/or is "immunologically
reactive" to a
polypeptide of the invention if it reacts at a detectable level (within, for
example, an
ELISA assay) with the polypeptide, and does not react detectably with
unrelated
polypeptides under similar conditions.
Immunological binding, as used in this context, generally refers to the
non-covalent interactions of the type which occur between an immunoglobulin
molecule and an antigen for which the immunoglobulin is specific. The
strength, or
affinity of immunological binding interactions can be expressed in terms of
the
dissociation constant (Kd) of the interaction, wherein a smaller Kd represents
a greater
2o affinity. Immunological binding properties of selected polypeptides can be
quantified
using methods well known in the art. One such method entails measuring the
rates of
antigen-binding site/antigen complex formation and dissociation, wherein those
rates
depend on the concentrations of the complex partners, the affinity of the
interaction, and
on geometric parameters that equally influence the rate in both directions.
Thus, both
the "on rate constant" (Ko") and the "off rate constant" (I~~.) can be
determined by
calculation of the concentrations and the actual rates of association and
dissociation.
The ratio of Ko~. /Ko~ enables cancellation of all parameters not related to
affinity, and is
thus equal to the dissociation constant Ka. See, generally, Davies et al.
(1990) Annual
Rev. Biochem. 59:439-473.
3o An "antigen-binding site," or "binding portion" of an antibody refers to
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the part of the immunoglobulin molecule that participates in antigen binding.
The
antigen binding site is formed by amino acid residues of the N-terminal
variable ("V")
regions of the heavy ("H") and light ("L") chains. Three highly divergent
stretches
within the V regions of the heavy and light chains are referred to as
"hypervariable
regions" which are interposed between more conserved flanking stretches known
as
"framework regions," or "FRs". Thus the term "FR" refers to amino acid
sequences
which are naturally found between and adjacent to hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable regions of a
light
chain and the three hypervariable regions of a heavy chain are disposed
relative to each
other in three dimensional space to form an antigen-binding surface. The
antigen-
binding surface is complementary to the three-dimensional surface of a bound
antigen,
and the three hypervariable regions of each of the heavy and light chains are
referred to
as "complementarity-determining regions," or "CDRs."
Binding agents may be further capable of differentiating between
patients with and without a cancer, such as ovarian cancer, using the
representative
assays provided herein. For example, antibodies or other binding agents that
bind to a
tumor protein will preferably generate a signal indicating the presence of a
cancer in at
least about 20% of patients with the disease, more preferably at least about
30% of
patients. Alternatively, or in addition, the antibody will generate a negative
signal
2o indicating the absence of the disease in at least about 90% of individuals
without the
cancer. To determine whether a binding agent satisfies this requirement,
biological
samples (e.g., blood, sera, sputum, urine and/or tumor biopsies) from patients
with and
without a cancer (as determined using standard clinical tests) may be assayed
as
described herein for the presence of polypeptides that bind to the binding
agent.
Preferably, a statistically significant number of samples with and without the
disease
will be assayed. Each binding agent should satisfy the above criteria;
however, those of
ordinary skill in the art will recognize that binding agents may be used in
combination
to improve sensitivity.
Any agent that satisfies the above requirements may be a binding agent.
3o For example, a binding agent may be a ribosome, with or without a peptide
component,
an RNA molecule or a polypeptide. In a preferred embodiment, a binding agent
is an
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antibody or an antigen-binding fragment thereof. Antibodies may be prepared by
any of
a variety of techniques known to those of ordinary skill in the art. See,
e.g., Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In
general, antibodies can be produced by cell culture techniques, including the
generation
of monoclonal antibodies as described herein, or via transfection of antibody
genes into
suitable bacterial or mammalian cell hosts, in order to allow for the
production of
recombinant antibodies. In one technique, an immunogen comprising the
polypeptide is
initially injected into any of a wide variety of mammals (e.g., mice, rats,
rabbits, sheep
or goats). In this step, the polypeptides of this invention may serve as the
immunogen
to without modification. Alternatively, particularly for relatively short
polypeptides, a
superior immune response may be elicited if the polypeptide is joined to a
carrier
protein, such as bovine serum albumin or keyhole limpet hemocyanin. The
immunogen
is injected into the animal host, preferably according to a predetermined
schedule
incorporating one or more booster immunizations, and the animals are bled
periodically.
Polyclonal antibodies specific for the polypeptide may then be purified from
such
antisera by, for example, affinity chromatography using the polypeptide
coupled to a
suitable solid support.
Monoclonal antibodies specific for an antigenic polypeptide of interest
may be prepared, for example, using the technique of Kohler and Milstein, Eur.
J.
2o Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods
involve
the preparation of immortal cell lines capable of producing antibodies having
the
desired specificity (i.e., reactivity with the polypeptide of interest). Such
cell lines may
be produced, for example, from spleen cells obtained from an animal immunized
as
described above. The spleen cells are then immortalized by, for example,
fusion with a
myeloma cell fusion partner, preferably one that is syngeneic with the
immunized
animal. A variety of fusion techniques may be employed. For example, the
spleen cells
and myeloma cells may be combined with a nonionic detergent for a few minutes
and
then plated at low density on a selective medium that supports the growth of
hybrid
cells, but not myeloma cells. A preferred selection technique uses HAT
(hypoxanthine,
3o aminopterin, thymidine) selection. After a sufficient time, usually about 1
to 2 weeks,
colonies of hybrids are observed. Single colonies are selected and their
culture
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supernatants tested for binding activity against the polypeptide. Hybridomas
having
high reactivity and specificity are preferred.
Monoclonal antibodies may be isolated from the supernatants of growing
hybridoma colonies. In addition, various techniques may be employed to enhance
the
yield, such as injection of the hybridoma cell line into the peritoneal cavity
of a suitable
vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested
from
the ascites fluid or the blood. Contaminants may be removed from the
antibodies by
conventional techniques, such as chromatography, gel filtration,
precipitation, and
extraction. The polypeptides of this invention may be used in the purification
process
1 o in, for example, an affinity chromatography step.
A number of therapeutically useful molecules are known in the art which
comprise antigen-binding sites that are capable of exhibiting immunological
binding
properties of an antibody molecule. The proteolytic enzyme papain
preferentially
cleaves IgG molecules to yield several fragments, two of which (the "F(ab)"
fragments)
t 5 each comprise a covalent heterodimer that includes an intact antigen-
binding site. The
enzyme pepsin is able to cleave IgG molecules to provide several fragments,
including
the "F(ab')2 " fragment which comprises both antigen-binding sites. An "Fv"
fragment
can be produced by preferential proteolytic cleavage of an IgM, and on rare
occasions
IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly
2o derived using recombinant techniques known in the art. The Fv fragment
includes a
non-covalent V,,:: VL heterodimer including an antigen-binding site which
retains much
of the antigen recognition and binding capabilities of the native antibody
molecule.
mbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al.
(1976)
Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.
25 A single chain Fv ("sFv") polypeptide is a covalently linked VH::VL
heterodimer which is expressed from a gene fusion including VH and V~ encoding
genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat.
Acad. Sci.
USA 85(16):5879-5883. A number of methods have been described to discern
chemical
structures for converting the naturally aggregated--but chemically separated--
light and
3o heavy polypeptide chains from an antibody V region into an sFv molecule
which will
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fold into a three dimensional structure substantially similar to the structure
of an
antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to
Huston et al.;
and U.S. Pat. No. 4,946,778, to Ladner et al.
Each of the above-described molecules includes a heavy chain and a
light chain CDR set, respectively interposed between a heavy chain and a light
chain FR
set which provide support to the CDRS and define the spatial relationship of
the CDRs
relative to each other. As used herein, the term "CDR set" refers to the three
hypervariable regions of a heavy or light chain V region. Proceeding from the
N-
terminus of a heavy or light chain, these regions are denoted as "CDR1,"
"CDR2," and
"CDR3" respectively. An antigen-binding site, therefore, includes six CDRs,
comprising the CDR set from each of a heavy and a light chain V region. A
polypeptide
comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) is referred to herein as
a
"molecular recognition unit." Crystallographic analysis of a number of antigen-
antibody
complexes has demonstrated that the amino acid residues of CDRs form extensive
contact with bound antigen, wherein the most extensive antigen contact is with
the
heavy chain CDR3. Thus, the molecular recognition units are primarily
responsible for
the specificity of an antigen-binding site.
As used herein, the term "FR set" refers to the four flanking amino acid
sequences which frame the CDRs of a CDR set of a heavy or light chain V
region.
2o Some FR residues may contact bound antigen; however, FRs are primarily
responsible
for folding the V region into the antigen-binding site, particularly the FR
residues
directly adjacent to the CDRS. Within FRs, certain amino residues and certain
structural
features are very highly conserved. In this regard, all V region sequences
contain an
internal disulfide loop of around 90 amino acid residues. When the V regions
fold into a
binding-site, the CDRs are displayed as projecting loop motifs which form an
antigen-
binding surface. It is generally recognized that there are conserved
structural regions of
FRs which influence the folded shape of the CDR loops into certain "canonical"
structures--regardless, of the precise CDR amino acid sequence. Further,
certain FR
residues are known to participate in non-covalent interdomain contacts which
stabilize
3o the interaction of the antibody heavy and light chains.
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A number of "humanized" antibody molecules comprising an antigen-
binding site derived from a non-human immunoglobulin have been described,
including
chimeric antibodies having rodent V regions and their associated CDRs fused to
human
constant domains (Winter et al. (1991) Nature 349:293-299; Lobuglio et al.
(1989)
Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-
4538; and Brown et al. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted
into a
human supporting FR prior to fusion with an appropriate human antibody
constant
domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988)
Science
239:1534-1536; and Jones et al. (1986) Nature 321:522-525), and rodent CDRs
to supported by recombinantly veneered rodent FRs (European Patent Publication
No.
519,596, published Dec. 23, 1992). These "humanized" molecules are designed to
minimize unwanted immunological response toward rodent antihuman antibody
molecules which limits the duration and effectiveness of therapeutic
applications of
those moieties in human recipients.
As used herein, the terms "veneered FRs" and "recombinantly veneered
FRs" refer to the selective replacement of FR residues from, e.g., a rodent
heavy or light
chain V region, with human FR residues in order to provide a xenogeneic
molecule
comprising an antigen-binding site which retains substantially all of the
native FR
polypeptide folding structure. Veneering techniques are based on the
understanding that
2o the ligand binding characteristics of an antigen-binding site are
determined primarily by
the structure and relative disposition of the heavy and light chain CDR sets
within the
antigen-binding surface. Davies et al. (1990) Ann. Rev. Biochem. 59:439-473.
Thus,
antigen binding specificity can be preserved in a humanized antibody only
wherein the
CDR structures, their interaction with each other, and their interaction with
the rest of
the V region domains are carefully maintained. By using veneering techniques,
exterior
(e.g., solvent-accessible) FR residues which are readily encountered by the
immune
system are selectively replaced with human residues to provide a hybrid
molecule that
comprises either a weakly immunogenic, or substantially non-immunogenic
veneered
surface.
The process of veneering makes use of the available sequence data for
human antibody variable domains compiled by Kabat et al., in Sequences of
Proteins of
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Immunological Interest, 4th ed., (U.S. Dept. of Health and Human Services,
U.S.
Government Printing Office, 1987), updates to the Kabat database, and other
accessible
U.S. and foreign databases (both nucleic acid and protein). Solvent
accessibilities of V
region amino acids can be deduced from the known three-dimensional structure
for
human and marine antibody fragments. There are two general steps in veneering
a
marine antigen-binding site. Initially, the FRs of the variable domains of an
antibody
molecule of interest are compared with corresponding FR sequences of human
variable
domains obtained from the above-identified sources. The most homologous human
V
regions are then compared residue by residue to corresponding marine amino
acids. The
to residues in the marine FR which differ from the human counterpart are
replaced by the
residues present in the human moiety using recombinant techniques well known
in the
art. Residue switching is only carried out with moieties which are at least
partially
exposed (solvent accessible), and care is exercised in the replacement of
amino acid
residues which may have a significant effect on the tertiary structure of V
region
domains, such as proline, glycine and charged amino acids.
In this manner, the resultant "veneered" marine antigen-binding sites are
thus designed to retain the marine CDR residues, the residues substantially
adjacent to
the CDRs, the residues identified as buried or mostly buried (solvent
inaccessible), the
residues believed to participate in non-covalent (e.g., electrostatic and
hydrophobic)
2o contacts between heavy and light chain domains, and the residues from
conserved
structural regions of the FRs which are believed to influence the "canonical"
tertiary
structures of the CDR loops. These design criteria are then used to prepare
recombinant
nucleotide sequences which combine the CDRs of both the heavy and light chain
of a
marine antigen-binding site into human-appearing FRs that can be used to
transfect
mammalian cells for the expression of recombinant human antibodies which
exhibit the
antigen specificity of the marine antibody molecule.
In another embodiment of the invention, monoclonal antibodies of the
present invention may be coupled to one or more therapeutic agents. Suitable
agents in
this regard include radionuclides, differentiation inducers, drugs, toxins,
and derivatives
3o thereof. Preferred radionuclides include 9°Y, ~z3I, 'zsl, 's'I,
'86Re, 'ggRe, z"At, and z'zBi.
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Preferred drugs include methotrexate, and pyrimidine and purine analogs.
Preferred
differentiation inducers include phorbol esters and butyric acid. Preferred
toxins
include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas
exotoxin,
Shigella toxin, and pokeweed antiviral protein.
A therapeutic agent may be coupled (e.g., covalently bonded) to a
suitable monoclonal antibody either directly or indirectly (e.g., via a linker
group). A
direct reaction between an agent and an antibody is possible when each
possesses a
substituent capable of reacting with the other. For example, a nucleophilic
group, such
as an amino or sulfhydryl group, on one may be capable of reacting with a
carbonyl-
1 o containing group, such as an anhydride or an acid halide, or with an alkyl
group
containing a good leaving group (e.g., a halide) on the other.
Alternatively, it may be desirable to couple a therapeutic agent and an
antibody via a linker group. A linker group can function as a spacer to
distance an
antibody from an agent in order to avoid interference with binding
capabilities. A
linker group can also serve to increase the chemical reactivity of a
substituent on an
agent or an antibody, and thus increase the coupling efficiency. An increase
in
chemical reactivity may also facilitate the use of agents, or functional
groups on agents,
which otherwise would not be possible.
It will be evident to those skilled in the art that a variety of bifunctional
or polyfunctional reagents, both homo- and hetero-functional (such as those
described
in the catalog of the Pierce Chemical Co., Rockford, IL), may be employed as
the linker
group. Coupling may be effected, for example, through amino groups, carboxyl
groups,
sulfhydryl groups or oxidized carbohydrate residues. There are numerous
references
describing such methodology, e.g., U.S. Patent No. 4,671,958, to Rodwell et
al.
Where a therapeutic agent is more potent when free from the antibody
portion of the immunoconjugates of the present invention, it may be desirable
to use a
linker group which is cleavable during or upon internalization into a cell. A
number of
different cleavable linker groups have been described. The mechanisms for the
intracellular release of an agent from these linker groups include cleavage by
reduction
of a disulfide bond (e.g., U.S. Patent No. 4,489,710, to Spider), by
irradiation of a
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photolabile bond (e.g., U.S. Patent No. 4,625,014, to Senter et al.), by
hydrolysis of
derivatized amino acid side chains (e.g., U.S. Patent No. 4,638,045, to Kohn
et al.), by
serum complement-mediated hydrolysis (e.g., U.S. Patent No. 4,671,958, to
Rodwell
et al.), and acid-catalyzed hydrolysis (e.g., U.S. Patent No. 4,569,789, to
Blattler et al.).
It may be desirable to couple more than one agent to an antibody. In one
embodiment, multiple molecules of an agent are coupled to one antibody
molecule. In
another embodiment, more than one type of agent may be coupled to one
antibody.
Regardless of the particular embodiment, immunoconjugates with more than one
agent
may be prepared in a variety of ways. For example, more than one agent may be
1 o coupled directly to an antibody molecule, or linkers that provide multiple
sites for
attachment can be used. Alternatively, a carrier can be used.
A carrier may bear the agents in a variety of ways, including covalent
bonding either directly or via a linker group. Suitable carriers include
proteins such as
albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides and
polysaccharides
such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.). A
carrier may
also bear an agent by noncovalent bonding or by encapsulation, such as within
a
liposome vesicle (e.g., U.S. Patent Nos. 4,429,008 and 4,873,088). Carriers
specific for
radionuclide agents include radiohalogenated small molecules and chelating
compounds. For example, U.S. Patent No. 4,735,792 discloses representative
radiohalogenated small molecules and their synthesis. A radionuclide chelate
may be
formed from chelating compounds that include those containing nitrogen and
sulfur
atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
For
example, U.S. Patent No. 4,673,562, to Davison et al. discloses representative
chelating
compounds and their synthesis.
T CELLS COMPOSITIONS
The present invention, in another aspect, provides T cells specific for a
tumor polypeptide disclosed herein, or for a variant or derivative thereof.
Such cells
may generally be prepared in vitro or ex vivo, using standard procedures. For
example,
3o T cells may be isolated from bone marrow, peripheral blood, or a fraction
of bone
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marrow or peripheral blood of a patient, using a commercially available cell
separation
system, such as the IsolexTM System, available from Nexell Therapeutics, Inc.
(Irvine,
CA; see also U.S. Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO
89/06280; WO
91/16116 and WO 92/07243). Alternatively, T cells may be derived from related
or
unrelated humans, non-human mammals, cell lines or cultures.
T cells may be stimulated with a polypeptide, polynucleotide encoding a
polypeptide and/or an antigen presenting cell (APC) that expresses such a
polypeptide.
Such stimulation is performed under conditions and for a time sufficient to
permit the
generation of T cells that are specific for the polypeptide of interest.
Preferably, a
1 o tumor polypeptide or polynucleotide of the invention is present within a
delivery
vehicle, such as a microsphere, to facilitate the generation of specific T
cells.
T cells are considered to be specific for a polypeptide of the present
invention if the T cells specifically proliferate, secrete cytokines or kill
target cells
coated with the polypeptide or expressing a gene encoding the polypeptide. T
cell
specificity may be evaluated using any of a variety of standard techniques.
For
example, within a chromium release assay or proliferation assay, a stimulation
index of
more than two fold increase in lysis and/or proliferation, compared to
negative controls,
indicates T cell specificity. Such assays may be performed, for example, as
described in
Chen et al., Cancer Res. 54:1065-1070, 1994. Alternatively, detection of the
2o proliferation of T cells may be accomplished by a variety of known
techniques. For
example, T cell proliferation can be detected by measuring an increased rate
of DNA
synthesis (e.g., by pulse-labeling cultures of T cells with tritiated
thymidine and
measuring the amount of tritiated thymidine incorporated into DNA). Contact
with a
tumor polypeptide (100 ng/ml - 100 ~g/ml, preferably' 200 ng/ml - 25 ~g/ml)
for 3 - 7
days will typically result in at least a two fold increase in proliferation of
the T cells.
Contact as described above for 2-3 hours should result in activation of the T
cells, as
measured using standard cytokine assays in which a two fold increase in the
level of
cytokine release (e.g., TNF or IFN-y) is indicative of T cell activation (see
Coligan et
al., Current Protocols in Immunology, vol. l, Wiley Interscience (Greene
1998)). T
3o cells that have been activated in response to a tumor polypeptide,
polynucleotide or
polypeptide-expressing APC may be CD4+ and/or CD8+. Tumor polypeptide-specific
T
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cells may be expanded using standard techniques. Within preferred embodiments,
the T
cells are derived from a patient, a related donor or an unrelated donor, and
are
administered to the patient following stimulation and expansion.
For therapeutic purposes, CD4+ or CD8+ T cells that proliferate in
response to a tumor polypeptide, polynucleotide or APC can be expanded in
number
either in vitro or in vivo. Proliferation of such T cells in vitro may be
accomplished in a
variety of ways. For example, the T cells can be re-exposed to a tumor
polypeptide, or
a short peptide corresponding to an immunogenic portion of such a polypeptide,
with or
without the addition of T cell growth factors, such as interleukin-2, and/or
stimulator
to cells that synthesize a tumor polypeptide. Alternatively, one or more T
cells that
proliferate in the presence of the tumor polypeptide can be expanded in number
by
cloning. Methods for cloning cells are well known in the art, and include
limiting
dilution.
PHARMACEUTICAL COMPOSITIONS
In additional embodiments, the present invention concerns formulation
of one or more of the polynucleotide, polypeptide, T-cell and/or antibody
compositions
disclosed herein in pharmaceutically-acceptable solutions for administration
to a cell or
an animal, either alone, or in combination with one or more other modalities
of therapy.
2o It will be understood that, if desired, a composition as disclosed herein
may be administered in combination with other agents as well, such as, e.g.,
other
proteins or polypeptides or various pharmaceutically-active agents. In fact,
there is
virtually no limit to other components that may also be included, given that
the
additional agents do not cause a significant adverse effect upon contact with
the target
cells or host tissues. The compositions may thus be delivered along with
various other
agents as required in the particular instance. Such compositions may be
purified from
host cells or other biological sources, or alternatively may be chemically
synthesized as
described herein. Likewise, such compositions may further comprise substituted
or
derivatized RNA or DNA compositions.
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Therefore, in another aspect of the present invention, pharmaceutical
compositions are provided comprising one or more of the polynucleotide,
polypeptide,
antibody, and/or T-cell compositions described herein in combination with a
physiologically acceptable carrier. In certain preferred embodiments, the
pharmaceutical compositions of the invention comprise immunogenic
polynucleotide
and/or polypeptide compositions of the invention for use in prophylactic and
theraputic
vaccine applications. Vaccine preparation is generally described in, for
example, M.F.
Powell and M.J. Newman, eds., "Vaccine Design (the subunit and adjuvant
approach),"
Plenum Press (NY, 1995). Generally, such compositions will comprise one or
more
to polynucleotide and/or polypeptide compositions of the present invention in
combination
with one or more immunostimulants.
It will be apparent that any of the pharmaceutical compositions described
herein can contain pharmaceutically acceptable salts of the polynucleotides
and
polypeptides of the invention. Such salts can be prepared, for example, from
~ 5 pharmaceutically acceptable non-toxic bases, including organic bases
(e.g., salts of
primary, secondary and tertiary amines and basic amino acids) and inorganic
bases
(e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).
In another embodiment, illustrative immunogenic compositions, e.g.,
vaccine compositions, of the present invention comprise DNA encoding one or
more of
2o the polypeptides as described above, such that the polypeptide is generated
in situ. As
noted above, the polynucleotide may be administered within any of a variety of
delivery
systems known to those of ordinary skill in the art. Indeed, numerous gene
delivery
techniques are well known in the art, such as those described by Rolland,
Crit. Rev.
Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein.
25 Appropriate polynucleotide expression systems will, of course, contain the
necessary
regulatory DNA regulatory sequences for expression in a patient (such as a
suitable
promoter and terminating signal). Alternatively, bacterial delivery systems
may involve
the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that
expresses an
immunogenic portion of the polypeptide on its cell surface or secretes such an
epitope.
3o Therefore, in certain embodiments, polynucleotides encoding
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immunogenic polypeptides described herein are introduced into suitable
mammalian
host cells for expression using any of a number of known viral-based systems.
In one
illustrative embodiment, retroviruses provide a convenient and effective
platform for
gene delivery systems. A selected nucleotide sequence encoding a polypeptide
of the
'present invention can be inserted into a vector and packaged in retroviral
particles using
techniques known in the art. The recombinant virus can then be isolated and
delivered
to a subject. A number of illustrative retroviral systems have been described
(e.g., U.S.
Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller,
A. D.
(1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852;
Burns
l0 et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and
Temin
(1993) Cur. Opin. Genet. Develop. 3:102-109.
In addition, a number of illustrative adenovirus-based systems have also
been described. Unlike retroviruses which integrate into the host genome,
adenoviruses
persist extrachromosomally thus minimizing the risks associated with
insertional
mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al.
(1993) J.
Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729;
Seth et
al. (1994) J: Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;
Berkner, K. L.
(1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy
4:461-
476).
2o Various adeno-associated virus (AAV) vector systems have also been
developed for polynucleotide delivery. AAV vectors can be readily constructed
using
techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and
5,139,941;
International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al.
(1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold
Spring
Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in
Biotechnology 3:533-
539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129;
Kotin, R. M. ( 1994) Human Gene Therapy 5:793-801; Shelling and Smith ( 1994)
Gene
Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.
Additional viral vectors useful for delivering the nucleic acid molecules
3o encoding polypeptides of the present invention by gene transfer include
those derived
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from the pox family of viruses, such as vaccinia virus and avian poxvirus. By
way of
example, vaccinia virus recombinants expressing the novel molecules can be
constructed as follows. The DNA encoding a polypeptide is first inserted into
an
appropriate vector so that it is adjacent to a vaccinia promoter and flanking
vaccinia
DNA sequences, such as the sequence encoding thymidine kinase (TK). This
vector is
then used to transfect cells which are simultaneously infected with vaccinia.
Homologous recombination serves to insert the vaccinia promoter plus the gene
encoding the polypeptide of interest into the viral genome. The resulting
TK<sup></sup>(-)
recombinant can be selected by culturing the cells in the presence of 5-
to bromodeoxyuridine and picking viral plaques resistant thereto.
A vaccinia-based infection/transfection system can be conveniently used
to provide for inducible, transient expression or coexpression of one or more
polypeptides described herein in host cells of an organism. In this particular
system,
cells are first infected in vitro with a vaccinia virus recombinant that
encodes the
bacteriophage T7 RNA polymerase. This polymerase displays exquisite
specificity in
that it only transcribes templates bearing T7 promoters. Following infection,
cells are
transfected with the polynucleotide or polynucleotides of interest, driven by
a T7
promoter. The polymerase expressed in the cytoplasm from the vaccinia virus
recombinant transcribes the transfected DNA into RNA which is then translated
into
2o polypeptide by the host translational machinery. The method provides for
high level,
transient, cytoplasmic production of large quantities of RNA and its
translation
products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)
87:6743-
6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.
Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,
can also be used to deliver the coding sequences of interest. Recombinant
avipox
viruses, expressing immunogens from mammalian pathogens, are known to confer
protective immunity when administered to non-avian species. The use of an
Avipox
vector is particularly desirable in human and other mammalian species since
members
of the Avipox genus can only productively replicate in susceptible avian
species and
3o therefore are not infective in mammalian cells. Methods for producing
recombinant
Avipoxviruses are known in the art and employ genetic recombination, as
described
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above with respect to the production of vaccinia viruses. See, e.g., WO
91/12882; WO
89/03429; and WO 92/03545.
Any of a number of alphavirus vectors can also be used for delivery of
polynucleotide compositions of the present invention, such as those vectors
described in
U.S. Patent Nos. 5,843,723; 6,015,686; 6,008,035 and 6,015,694. Certain
vectors based
on Venezuelan Equine Encephalitis (VEE) can also be used, illustrative
examples of
which can be found in U.S. Patent Nos. 5,505,947 and 5,643,576.
Moreover, molecular conjugate vectors, such as the adenovirus chimeric
vectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869 and
Wagner et
to al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for
gene delivery
under the invention.
Additional illustrative information on these and other known viral-based
delivery systems can be found, for example, in Fisher-Hoch et al., Proc. Natl.
Acad. Sci.
USA 86:317-321, 1989; Flexner et al., Ann. N Y. Acad. Sci. 569:86-103, 1989;
Flexner
et al., Vaccine 8:17-21, 1990; U.S. Patent Nos. 4,603,112, 4,769,330, and
5,017,487;
WO 89/01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO
91/02805;
Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,
1991;
Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al.,
Proc. Natl.
Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848,
1993;
2o and Guzman et al., Cir. Res. 73:1202-1207, 1993.
In certain embodiments, a polynucleotide may be integrated into the
genome of a target cell. This integration may be in the specific location and
orientation
via homologous recombination (gene replacement) or it may be integrated in a
random,
non-specific location (gene augmentation). In yet further embodiments, the
polynucleotide may be stably maintained in the cell as a separate, episomal
segment of
DNA. Such polynucleotide segments or "episomes" encode sequences sufficient to
permit maintenance and replication independent of or in synchronization with
the host
cell cycle. The mariner in which the expression construct is delivered to a
cell and
where in the cell the polynucleotide remains is dependent on the type of
expression
3o construct employed.
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In another embodiment of the invention, a polynucleotide is
administered/delivered as "naked" DNA, for example as described in Ulmer et
al.,
Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692,
1993.
The uptake of naked DNA may be increased by coating the DNA onto biodegradable
beads, which are efficiently transported into the cells.
In still another embodiment, a composition of the present invention can
be delivered via a particle bombardment approach, many of which have been
described.
In one illustrative example, gas-driven particle acceleration can be achieved
with
devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford,
UK)
1o and Powderject Vaccines Inc. (Madison, WI), some examples of which are
described in
U.S. Patent Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No.
0500
799. This approach offers a needle-free delivery approach wherein a dry powder
formulation of microscopic particles, such as polynucleotide or polypeptide
particles,
are accelerated to high speed within a helium gas jet generated by a hand held
device,
propelling the particles into a target tissue of interest.
In a related embodiment, other devices and methods that may be useful
for gas-driven needle-less injection of compositions of the present invention
include
those provided by Bioject, Inc. (Portland, OR), some examples of which are
described
in U.S. Patent Nos. 4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163;
5,520,639
2o and 5,993,412.
According to another embodiment, the pharmaceutical compositions
described herein will comprise one or more immunostimulants in addition to the
immunogenic polynucleotide, polypeptide, antibody, T-cell and/or APC
compositions
of this invention. An immunostimulant refers to essentially any substance that
enhances
or potentiates an immune response (antibody and/or cell-mediated) to an
exogenous
antigen. One preferred type of immunostimulant comprises an adjuvant. Many
adjuvants contain a substance designed to protect the antigen from rapid
catabolism,
such as aluminum hydroxide or mineral oil, and a stimulator of immune
responses, such
as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived
proteins.
3o Certain adjuvants are commercially available as, for example, Freund's
Incomplete
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Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck
Adjuvant
65 (Merck and Company, Inc., Rahway, N~; AS-2 (SmithKline Beecham,
Philadelphia,
PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum
phosphate;
salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine;
acylated
sugars; cationically or anionically derivatized polysaccharides;
polyphosphazenes;
biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such
as
GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be
used as
adj uvants.
Within certain embodiments of the invention, the adjuvant composition
l0 is preferably one that induces an immune response predominantly of the Thl
type.
High levels of Thl-type cytokines (e.g., IFN-y, TNFa, IL-2 and IL-12) tend to
favor the
induction of cell mediated immune responses to an administered antigen. In
contrast,
high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to
favor the
induction of humoral immune responses. Following application of a vaccine as
provided herein, a patient will support an immune response that includes Thl-
and Th2-
type responses. Within a preferred embodiment, in which a response is
predominantly
Thl-type, the level of Thl-type cytokines will increase to a greater extent
than the level
of Th2-type cytokines. The levels of these cytokines may be readily assessed
using
standard assays. For a review of the families of cytokines, see Mosmann and
Coffman,
2o Ann. Rev. Immunol. 7:145-173, 1989.
Certain preferred adjuvants for eliciting a predominantly Thl-type
response include, for example, a combination of monophosphoryl lipid A,
preferably 3-
de-O-acylated monophosphoryl lipid A, together with an aluminum salt. MPL~
adjuvants are available from Corixa Corporation (Seattle, WA; see, for
example, US
Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing
oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a
predominantly Thl response. Such oligonucleotides are well known and are
described,
for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and
5,856,462. Immunostimulatory DNA sequences are also described, for example, by
3o Sato et al., Science 273:352, 1996. Another preferred adjuvant comprises a
saponin,
such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila
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Biopharmaceuticals Inc., Framingham, MA); Escin; Digitonin; or Gypsophila or
Chenopodium quinoa saponins . Other preferred formulations include more than
one
saponin in the adjuvant combinations of the present invention, for example
combinations of at least two of the following group comprising QS21, QS7, Quil
A, (3-
escin, or digitonin.
Alternatively the saponin formulations may be combined with vaccine
vehicles composed of chitosan or other polycationic polymers, polylactide and
polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer
matrix,
particles composed of polysaccharides or chemically modified polysaccharides,
liposomes and lipid-based particles, particles composed of glycerol
monoesters, etc.
The saponins may also be formulated in the presence of cholesterol to form
particulate
structures such as liposomes or ISCOMs. Furthermore, the saponins may be
formulated
together with a polyoxyethylene ether or ester, in either a non-particulate
solution or
suspension, or in a particulate structure such as a paucilamelar liposome or
ISCOM.
~ 5 The saponins may also be formulated with excipients such as CarbopolR to
increase
viscosity, or may be formulated in a dry powder form with a powder excipient
such as
lactose.
In one preferred embodiment, the adjuvant system includes the
combination of a monophosphoryl lipid A and a saponin derivative, such as the
2o combination of QS21 and 3D-MPL~ adjuvant, as described in WO 94/00153, or a
less
reactogenic composition where the QS21 is quenched with cholesterol, as
described in
WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion
and
tocopherol. Another particularly preferred adjuvant formulation employing
QS21, 3D-
MPL~ adjuvant and tocopherol in an oil-in-water emulsion is described in WO
25 95/17210.
Another enhanced adjuvant system involves the combination of a CpG-
containing oligonucleotide and a saponin derivative particularly the
combination of
CpG and QS21 as disclosed in WO 00/09159. Preferably the formulation
additionally
comprises an oil in water emulsion and tocopherol.
Additional illustrative adjuvants for use in the pharmaceutical
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compositions of the invention include Montanide ISA 720 (Seppic, France), SAF
(Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS
series
of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham,
Rixensart,
Belgium), Detox (Enhanzyn~) (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton,
MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those
described
in pending U.S. Patent Application Serial Nos. 08/853,826 and 09/074,720, the
disclosures of which are incorporated herein by reference in their entireties,
and
polyoxyethylene ether adjuvants such as those described in WO 99/52549A1.
Other preferred adjuvants include adjuvant molecules of the general formula
(I):
~ o HO(CHZCHzO)~-A-R
Wherein, n is 1-50, A is a bond or -C(O)-, R is C,_SO alkyl or Phenyl C,_SO
alkyl.
One embodiment of the present invention consists of a vaccine
formulation comprising a polyoxyethylene ether of general formula (I), wherein
n is
between 1 and 50, preferably 4-24, most preferably 9; the R component is C,_SO
preferably C4-Czo alkyl and most preferably C,2 alkyl, and A is a bond. The
concentration of the polyoxyethylene ethers should be in the range 0.1-20%,
preferably
from 0.1-10%, and most preferably in the range 0.1-1%. Preferred
polyoxyethylene
ethers are selected from the following group: polyoxyethylene-9-lauryl ether,
polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,
polyoxyethylerie-4-
lauryl ether; polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl
ether.
Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in
the Merck
index (12'" edition: entry 7717). These adjuvant molecules are described in WO
99/52549.
The polyoxyethylene ether according to the general formula (I) above
z5 may, if desired, be combined with another adjuvant. For example, a
preferred adjuvant
combination is preferably with CpG as described in the pending UK patent
application
GB 9820956.2.
According to another embodiment of this invention, an immunogenic
composition described herein is delivered to a host via antigen presenting
cells (APCs),
3o such as dendritic cells, macrophages, B cells, monocytes and other cells
that may be
engineered to be efficient APCs. Such cells may, but need not, be genetically
modified
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to increase the capacity for presenting the antigen, to improve activation
and/or
maintenance of the T cell response, to have anti-tumor effects per se and/or
to be
immunologically compatible with the receiver (i. e., matched HLA haplotype).
APCs
may generally be isolated from any of a variety of biological fluids and
organs,
s including tumor and peritumoral tissues, and may be autologous, allogeneic,
syngeneic
or xenogeneic cells.
Certain preferred embodiments of the present invention use dendritic
cells or progenitors thereof as antigen-presenting cells. Dendritic cells are
highly potent
APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown
to
1o be effective as a physiological adjuvant for eliciting prophylactic or
therapeutic
antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999).
In
general, dendritic cells may be identified based on their typical shape
(stellate in situ,
with marked cytoplasmic processes (dendrites) visible in vitro), their ability
to take up,
process and present antigens with high efficiency and their ability to
activate naive T
15 cell responses: Dendritic cells may, of course, be engineered to express
specific cell-
surface receptors or ligands that are not commonly found on dendritic cells in
vivo or ex
vivo, and such modified dendritic cells are contemplated by the present
invention. As
an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic
cells (called
exosomes) may be used within a vaccine (see Zitvogel et al., Nature Med. 4:594-
600,
20 1998).
Dendritic cells and progenitors may be obtained from peripheral blood,
bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells,
lymph
nodes; spleen, skin, umbilical cord blood or any other suitable tissue or
fluid. For
example, dendritic cells may be differentiated ex vivo by adding a combination
of
25 cytokines such as GM-CSF, IL-4, IL-13 and/or TNFa to cultures of monocytes
harvested from peripheral blood. Alternatively, CD34 positive cells harvested
from
peripheral blood, umbilical cord blood or bone marrow may be differentiated
into
dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3,
TNFa,
CD40 ligand, LPS, flt3 ligand and/or other compounds) that induce
differentiation,
3o maturation and proliferation of dendritic cells.
Dendritic cells are conveniently categorized as "immature" and "mature"
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cells, which allows a simple way to discriminate between two well
characterized
phenotypes. However, this nomenclature should not be construed to exclude all
possible intermediate stages of differentiation. Immature dendritic cells are
characterized as APC with a high capacity for antigen uptake and processing,
which
correlates with the high expression of Fcy receptor and mannose receptor. The
mature
phenotype is typically characterized by a lower expression of these markers,
but a high
expression of cell surface molecules responsible for T cell activation such as
class I and
class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory
molecules
(e.g., CD40, CD80, CD86 and 4-1BB).
to APCs may generally be transfected with a polynucleotide of the
invention (or portion or other variant thereof) such that the encoded
polypeptide, or an
immunogenic portion thereof, is expressed on the cell surface. Such
transfection may
take place ex vivo, and a pharmaceutical composition comprising such
transfected cells
may then be used for therapeutic purposes, as described herein. Alternatively,
a gene
delivery vehicle that targets a dendritic or other antigen presenting cell may
be
administered to a patient, resulting in transfection that occurs in vivo. In
vivo and ex
vivo transfection of dendritic cells, for example, may generally be performed
using any
methods known in the art, such as those described in WO 97/24447, or the gene
gun
approach described by Mahvi et al., Immunology and cell Biology 75:456-460,
1997.
2o Antigen loading of dendritic cells may be achieved by incubating dendritic
cells or
progenitor cells with the tumor polypeptide, DNA (naked or within a plasmid
vector) or
RNA; or with antigen-expressing recombinant bacterium or viruses (e.g.,
vaccinia,
fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide
may be
covalently conjugated to an immunological partner that provides T cell help
(e.g., a
carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-
conjugated
immunological partner, separately or in the presence of the polypeptide.
While any suitable carrier known to those of ordinary skill in the art may
be employed in the pharmaceutical compositions of this invention, the type of
carrier
will typically vary depending on the mode of administration. Compositions of
'the
3o present invention may be formulated for any appropriate manner of
administration,
including for example, topical, oral, nasal, mucosal, intravenous,
intracranial,
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intraperitoneal, subcutaneous and intramuscular administration.
Carriers for use within such pharmaceutical compositions are
biocompatible, and may also be biodegradable. In certain embodiments, the
formulation preferably provides a relatively constant level of active
component release.
s In other embodiments, however, a more rapid rate of release immediately upon
administration may be desired. The formulation of such compositions is well
within the
level of ordinary skill in the art using known techniques. Illustrative
carriers useful in
this regard include microparticles of poly(lactide-co-glycolide),
polyacrylate, latex,
starch, cellulose, dextran and the like. Other illustrative delayed-release
carriers
1o include supramolecular biovectors, which comprise a non-liquid hydrophilic
core (e.g.,
a cross-linked polysaccharide or oligosaccharide) and, optionally, an external
layer
comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S.
Patent No.
5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The
amount of active compound contained within a sustained release formulation
depends
~ 5 upon the site of implantation, the rate and expected duration of release
and the nature of
the condition to be treated or prevented.
In another illustrative embodiment, biodegradable microspheres (e.g.,
polylactate polyglycolate) are employed as carriers for the compositions of
this
invention. Suitable biodegradable microspheres are disclosed, for example, in
U.S.
2o Patent Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883;
5,853,763;
5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier
systems.
such as described in WO/99 40934, and references cited therein, will also be
useful for
many applications. Another illustrative carrier/delivery system employs a
carrier
comprising particulate-protein complexes, such as those described in U.S.
Patent No.
25 5,928,647, which are capable of inducing a class I-restricted cytotoxic T
lymphocyte
responses in a host.
The pharmaceutical compositions of the invention will often further
comprise one or more buffers (e.g., neutral buffered saline or phosphate
buffered
saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans),
mannitol, proteins,
3o polypeptides or amino acids such as glycine, antioxidants, bacteriostats,
chelating
agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide),
solutes that
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render the formulation isotonic, hypotonic or weakly hypertonic with the blood
of a
recipient, suspending agents, thickening agents and/or preservatives.
Alternatively,
compositions of the present invention may be formulated as a lyophilizate.
The pharmaceutical compositions described herein may be presented in
unit-dose or multi-dose containers, such as sealed ampoules or vials. Such
containers
are typically sealed in such a way to preserve the sterility and stability of
the
formulation until use. In general, formulations may be stored as suspensions,
solutions
or emulsions in oily or aqueous vehicles. Alternatively, a pharmaceutical
composition
may be stored in a freeze-dried condition requiring only the addition of a
sterile liquid
carrier immediately prior to use.
The development of suitable dosing and treatment regimens for using the
particular compositions described herein in a variety of treatment regimens,
including
e.g., oral, parenteral, intravenous, intranasal, and intramuscular
administration and
formulation, is well known in the art, some of which are briefly discussed
below for
general purposes of illustration.
In certain applications, the pharmaceutical compositions disclosed herein
may be delivered via oral administration to an animal. As such, these
compositions
may be formulated with an inert diluent or with an assimilable edible carrier,
or they
may be enclosed in hard- or soft-shell gelatin capsule, or they may be
compressed into
2o tablets, or they may be incorporated directly with the food of the diet.
The active compounds may even be incorporated with excipients and
used in the form of ingestible tablets, buccal tables, troches, capsules,
elixirs,
suspensions, syrups, wafers, and the like (see, for example, Mathiowitz et
al., Nature
1997 Mar 27;386(6623):410-4; Hwang et al., Crit Rev Ther Drug Carrier Syst
1998;15(3):243-84; U. S. Patent 5,641,515; U. S. Patent 5,580,579 and U. S.
Patent
5,792,451 ). Tablets, troches, pills, capsules and the like may also contain
any of a
variety of additional components, for example, a binder, such as gum
tragacanth, acacia,
cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent,
such as corn starch, potato starch, alginic acid and the like; a lubricant,
such as
magnesium stearate; and a sweetening agent, such as sucrose, lactose or
saccharin may
be added or a flavoring agent, such as peppermint, oil of wintergreen, or
cherry
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flavoring. When the dosage unit form is a capsule, it may contain, in addition
to
materials of the above type, a liquid carrier. Various other materials may be
present as
coatings or to otherwise modify the physical form of the dosage unit. For
instance,
tablets, pills, or capsules may be coated with shellac, sugar, or both. Of
course, any
material used in preparing any dosage unit form should be pharmaceutically
pure and
substantially non-toxic in the amounts employed. In addition, the active
compounds
may be incorporated into sustained-release preparation and formulations.
Typically, these formulations will contain at least about 0.1 % of the
active compound or more, although the percentage of the active ingredients)
may, of
1o course, be varied and may conveniently be between about 1 or 2% and about
60% or
70% or more of the weight or volume of the total formulation. Naturally, the
amount of
active compounds) in each therapeutically useful composition may be prepared
is such
a way that a suitable dosage will be obtained in any given unit dose of the
compound.
Factors such as solubility, bioavailability, biological half life, route of
administration,
product shelf life, as well as other pharmacological considerations will be
contemplated
by one skilled in the art of preparing such pharmaceutical formulations, and
as such, a
variety of dosages and treatment regimens may be desirable.
For oral administration the compositions of the present invention may
alternatively be incorporated with one or more excipients in the form of a
mouthwash;
dentifrice, buccal tablet, oral spray, or sublingual orally-administered
formulation.
Alternatively, the active ingredient may be incorporated into an oral solution
such as
one containing sodium borate, glycerin and potassium bicarbonate, or dispersed
in a
dentifrice, or added in a therapeutically-effective amount to a composition
that may
include water, binders, abrasives, flavoring agents, foaming agents, and
humectants.
Alternatively the compositions may be fashioned into a tablet or solution form
that may
be placed under the tongue or otherwise dissolved in the mouth.
In certain circumstances it will be desirable to deliver the pharmaceutical
compositions disclosed herein parenterally, intravenously, intramuscularly, or
even
intraperitoneally. Such approaches are well known to the skilled artisan, some
of which
are further described, for example, in U. S. Patent 5,543,158; U. S. Patent
5,641,515 and
U. S. Patent 5,399,363. In certain embodiments, solutions of the active
compounds as
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free base or pharmacologically acceptable salts may be prepared in water
suitably
mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also
be
prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils.
Under ordinary conditions of storage and use, these preparations generally
will contain
a preservative to prevent the growth of microorganisms.
Illustrative pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersions (for example, see
U. S. Patent
5,466,468). In all cases the form must be sterile and must be fluid to the
extent that
to easy syringability exists. It must be stable under the conditions of
manufacture and
storage and must be preserved against the contaminating action of
microorganisms,
such as bacteria and fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (e.g., glycerol, propylene
glycol, and
liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or
vegetable
oils. Proper fluidity may be maintained, for example, by the use of a coating,
such as
lecithin, by the maintenance of the required particle size in the case of
dispersion and/or
by the use of surfactants. The prevention of the action of microorganisms can
be
facilitated by various antibacterial and antifungal agents, for example,
parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases,
it will be
preferable to include isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of the injectable compositions can be brought about by
the use in
the compositions of agents delaying absorption, for example, aluminum
monostearate
and gelatin.
In one embodiment, for parenteral administration in an aqueous solution,
the solution should be suitably buffered if necessary and the liquid diluent
first rendered
isotonic with sufficient saline or glucose. These particular aqueous solutions
are
especially suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal
administration. In this connection, a sterile aqueous medium that can be
employed will
be known to those of skill in the art in light of the present disclosure. For
example, one
3o dosage may be dissolved in 1 ml of isotonic NaCI solution and either added
to 1000 ml
of hypodermoclysis fluid or injected at the proposed site of infusion, (see
for example,
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"Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-
1580). Some variation in dosage will necessarily occur depending on the
condition of
the subject being treated. Moreover, for human administration, preparations
will of
course preferably meet sterility, pyrogenicity, and the general safety and
purity
standards as required by FDA Office of Biologics standards.
In another embodiment of the invention, the compositions disclosed
herein may be formulated in a neutral or salt form. Illustrative
pharmaceutically-acceptable salts include the acid addition salts (formed with
the free
amino groups of the protein) and which are formed with inorganic acids such
as, for
l0 example, hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic,
tartaric, mandelic, and the like. Salts formed with the free carboxyl groups
can also be
derived from inorganic bases such as, for example, sodium, potassium,
ammonium,
calcium, or ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation, solutions
will be
administered in a manner compatible with the dosage formulation and in such
amount
as is therapeutically effective.
The carriers can further comprise any and all solvents, dispersion media,
vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic
and absorption
delaying agents, buffers, carrier solutions, suspensions, colloids, and the
like. The use
of such media and agents for pharmaceutical active substances is well known in
the art.
Except insofar as any conventional media or agent is incompatible with the
active
ingredient, its use in the therapeutic compositions is contemplated.
Supplementary
active ingredients can also be incorporated into the compositions. The phrase
"pharmaceutically-acceptable" refers to molecular entities and compositions
that do not
produce an allergic or similar untoward reaction when administered to a human.
In certain embodiments, the pharmaceutical compositions may be
delivered by intranasal sprays, inhalation, and/or other aerosol delivery
vehicles.
Methods for delivering genes, nucleic acids, and peptide compositions directly
to the
lungs via nasal aerosol sprays has been described, e.g., in U. S. Patent
5,756,353 and U.
3o S. Patent 5,804,212. Likewise, the delivery of drugs using intranasal
microparticle
resins (Takenaga et al., J Controlled Release 1998 Mar 2;52(1-2):81-7) and
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lysophosphatidyl-glycerol compounds (U. S. Patent 5,725,871) are also well-
known in
the pharmaceutical arts. Likewise, illustrative transmucosal drug delivery in
the form of
a polytetrafluoroetheylene support matrix is described in U. S. Patent
5,780,045.
In certain embodiments, liposomes, nanocapsules, microparticles, lipid
particles, vesicles, and the like, are used for the introduction of the
compositions of the
present invention into suitable host cells/organisms. In particular, the
compositions of
the present invention may be formulated for delivery either encapsulated in a
lipid
particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
Alternatively,
compositions of the present invention can be bound, either covalently or non
to covalently, to the surface of such carrier vehicles.
The formation and use of liposome and liposome-like preparations as
potential drug carriers is generally known to those of skill in the art (see
for example,
Lasic, Trends Biotechnol 1998 Ju1;16(7):307-21; Takakura, Nippon Rinsho 1998
Mar;56(3):691-5; Chandran et al., Indian J Exp Biol. 1997 Aug;35(8):801-9;
Margalit,
Crit Rev Ther Drug Carrier Syst. 1995;12(2-3):233-61; U.S. Patent 5,567,434;
U.S.
Patent 5,552,157; U.S. Patent 5,565,213; U.S. Patent 5,738,868 and U.S. Patent
5,795,587, each specifically incorporated herein by reference in its
entirety).
Liposomes have been used successfully with a number of cell types that
are normally difficult to transfect by other procedures, including T cell
suspensions,
2o primary hepatocyte cultures and PC 12 cells (Renneisen et al., J Biol Chem.
1990 Sep
25;265(27):16337-42; Muller et al., DNA Cell Biol. 1990 Apr;9(3):221-9). In
addition,
liposomes are free of the DNA length constraints that are typical of viral-
based delivery
systems. Liposomes have been used effectively to introduce genes, various
drugs,
radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric
effectors and
the like, into a variety of cultured cell lines and animals. Furthermore, he
use of
liposomes does not appear to be associated with autoimmune responses or
unacceptable
toxicity after systemic delivery.
In certain embodiments, liposomes are formed from phospholipids that
are dispersed in an aqueous medium and spontaneously form multilamellar
concentric
3o bilayer vesicles (also termed multilamellar vesicles (MLVs).
Alternatively, in other embodiments, the invention provides for
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pharmaceutically-acceptable nanocapsule formulations of the compositions of
the
present invention. Nanocapsules can generally entrap compounds in a stable and
reproducible way (see, for example, Quintanar-Guerrero et al., Drug Dev Ind
Pharm.
1998 Dec;24(12):1113-28). To avoid side effects due to intracellular polymeric
overloading, such ultrafine particles (sized around 0.1 p.m) may be designed
using
polymers able to be degraded in vivo. Such particles can be made as described,
for
example, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20;
zur
Muhlen et al., Eur J Pharm Biopharm. 1998 Mar;45(2):149-55; Zambaux et al. J
Controlled Release. 1998 Jan 2;50(1-3):31-40; and U. S. Patent 5,145,684.
CANCER THERAPEUTIC METHODS
In further aspects of the present invention, the pharmaceutical
compositions described herein may be used for the treatment of cancer,
particularly for
the immunotherapy of ovarian cancer. Within such methods, the pharmaceutical
compositions described herein are administered to a patient, typically a warm-
blooded
animal, preferably a human. A patient may or may not be afflicted with cancer.
Accordingly, the above pharmaceutical compositions may be used to prevent the
development of a cancer or to treat a patient afflicted with a cancer.
Pharmaceutical
compositions and vaccines may be administered either prior to or following
surgical
2o removal of primary tumors and/or treatment such as administration of
radiotherapy or
conventional chemotherapeutic drugs. As discussed above, administration of the
pharmaceutical compositions may be by any suitable method, including
administration
by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal,
intradermal,
anal, vaginal, topical and oral routes.
Within certain embodiments, immunotherapy may be active
immunotherapy, in which treatment relies on the in vivo stimulation of the
endogenous
host immune system to react against tumors with the administration of immune
response-modifying agents (such as polypeptides and polynucleotides as
provided
herein). .
Within other embodiments, immunotherapy may be passive
immunotherapy, in which treatment involves the delivery of agents with
established
CA 02384499 2002-03-08
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tumor-immune reactivity (such as effector cells or antibodies) that can
directly or
indirectly mediate antitumor effects and does not necessarily depend on an
intact host
immune system. Examples of effector cells include T cells as discussed above,
T
lymphocytes (such as CD8+ cytotoxic T lymphocytes and CD4+ T-helper tumor-
s infiltrating lymphocytes), killer cells (such as Natural Killer cells and
lymphokine-
activated killer cells), B cells and antigen-presenting cells (such as
dendritic cells and
macrophages) expressing a polypeptide provided herein. T cell receptors and
antibody
receptors specific for the polypeptides recited herein may be cloned,
expressed and
transferred into other vectors or effector cells for adoptive immunotherapy.
The
to polypeptides provided herein may also be used to generate antibodies or
anti-idiotypic
antibodies (as described above and in U.S. Patent No. 4,918,164) for passive
immunotherapy.
Effector cells may generally be obtained in sufficient quantities for
adoptive immunotherapy by growth in vitro, as described herein. Culture
conditions for
15 expanding single antigen-specific effector cells to several billion in
number with
retention of antigen recognition in vivo are well known in the art. Such in
vitro culture
conditions typically use intermittent stimulation with antigen, often in the
presence of
cytokines (such as IL-2) and non-dividing feeder cells. As noted above,
immunoreactive polypeptides as provided herein may be used to rapidly expand
2o antigen-specific T cell cultures in order to generate a sufficient number
of cells for
immunotherapy. In particular, antigen-presenting cells, such as dendritic,
macrophage,
monocyte, fibroblast and/or B cells, may be pulsed with immunoreactive
polypeptides
or transfected with one or more polynucleotides using standard techniques well
known
in the art. For example, antigen-presenting cells can be transfected with a
25 polynucleotide having a promoter appropriate for increasing expression in a
recombinant virus or other expression system. Cultured effector cells for use
in therapy
must be able to grow and distribute widely, and to survive long term in vivo.
Studies
have shown that cultured effector cells can be induced to grow in vivo and to
survive
long term in substantial numbers by repeated stimulation with antigen
supplemented
3o with IL-2 (see, for example, Cheever et al., Immunological Reviews 157:177,
1997).
Alternatively, a vector expressing a polypeptide recited herein may be
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introduced into antigen presenting cells taken from a patient and clonally
propagated ex
vivo for transplant back into the same patient. Transfected cells may be
reintroduced
into the patient using any means known in the art, preferably in sterile form
by
intravenous, intracavitary, intraperitoneal or intratumor administration.
Routes and frequency of administration of the therapeutic compositions
described herein, as well as dosage, will vary from individual to individual,
and may be
readily established using standard techniques. In general, the pharmaceutical
compositions and vaccines may be administered by injection (e.g.,
intracutaneous,
intramuscular, intravenous or subcutaneous), intranasally (e.g., by
aspiration) or orally.
t o Preferably, between 1 and 10 doses may be administered over a 52 week
period.
Preferably, 6 doses are administered, at intervals of 1 month, and booster
vaccinations
may be given periodically thereafter. Alternate protocols may be appropriate
for
individual patients. A suitable dose is an amount of a compound that, when
administered as described above, is capable of promoting an anti-tumor immune
response, and is at least 10-50% above the basal (i.e., untreated) level. Such
response
can be monitored by measuring the anti-tumor antibodies in a patient or by
vaccine-
dependent generation of cytolytic effector cells capable of killing the
patient's tumor
cells in vitro. Such vaccines should also be capable of causing an immune
response that
leads to an improved clinical outcome (e.g., more frequent remissions,
complete or
2o partial or longer disease-free survival) in vaccinated patients as compared
to non-
vaccinated patients. In general, for pharmaceutical compositions and vaccines
comprising one or more polypeptides, the amount of each polypeptide present in
a dose
ranges from about 25 ~,g to 5 mg per kg of host. Suitable dose sizes will vary
with the
size of the patient, but will typically range from about 0.1 mL to about 5 mL.
In general, an appropriate dosage and treatment regimen provides the
active compounds) in an amount sufficient to provide therapeutic and/or
prophylactic
benefit. Such a response can be monitored by establishing an improved clinical
outcome (e.g., more frequent remissions, complete or partial, or longer
disease-free
survival) in treated patients as compared to non-treated patients. Increases
in
3o preexisting immune responses to a tumor protein generally correlate with an
improved
clinical outcome. Such immune responses may generally be evaluated using
standard
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proliferation, cytotoxicity or cytokine assays, which may be performed using
samples
obtained from a patient before and after treatment.
CANCER DETECTION AND DIAGNOSTIC COMPOSITIONS, METHODS AND KITS
s In general, a cancer may be detected in a patient based on the presence of
one or more ovarian tumor proteins and/or polynucleotides encoding such
proteins in a
biological sample (for example, blood, sera, sputum urine and/or tumor
biopsies)
obtained from the patient. In other words, such proteins may be used as
markers to
indicate the presence or absence of a cancer such as ovarian cancer. In
addition, such
1o proteins may be useful for the detection of other cancers. The binding
agents provided
herein generally permit detection of the level of antigen that binds to the
agent in the
biological sample. Polynucleotide primers and probes may be used to detect the
level
of mRNA encoding a tumor protein, which is also indicative of the presence or
absence
of a cancer. In general, a ovarian tumor sequence should be present at a level
that is at
15 least three fold higher in tumor tissue than in normal tissue
There are a variety of assay formats known to those of ordinary skill in
the art for using a binding agent to detect polypeptide markers in a sample.
See, e.g.,
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory,
1988. In general, the presence or absence of a cancer in a patient may be
determined by
20 (a) contacting a biological sample obtained from a patient with a binding
agent; (b)
detecting in the sample a level of polypeptide that binds to the binding
agent; and (c)
comparing the level of polypeptide with a predetermined cut-off value.
In a preferred embodiment, the assay involves the use of binding agent
immobilized on a solid support to bind to and remove the polypeptide from the
25 remainder of the sample. The bound polypeptide may then be detected using a
detection reagent that contains a reporter group and specifically binds to the
binding
agent/polypeptide complex. Such detection reagents may comprise, for example,
a
binding agent that specifically binds to the polypeptide or an antibody or
other agent
that specifically binds to the binding agent, such as an anti-immunoglobulin,
protein G,
3o protein A or a lectin. Alternatively, a competitive assay may be utilized,
in which a
polypeptide is labeled with a reporter group and allowed to bind to the
immobilized
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binding agent after incubation of the binding agent with the sample. The
extent to
which components of the sample inhibit the binding of the labeled polypeptide
to the
binding agent is indicative of the reactivity of the sample with the
immobilized binding
agent. Suitable polypeptides for use within such assays include full length
ovarian
tumor proteins and polypeptide portions thereof to which the binding agent
binds, as
described above.
The solid support may be any material known to those of ordinary skill
in the art to which the tumor protein may be attached. For example, the solid
support
may be a test well in a microtiter plate or a nitrocellulose or other suitable
membrane.
1o Alternatively, the support may be a bead or disc, such as glass,
fiberglass, latex or a
plastic material such as polystyrene or polyvinylchloride. The support may
also be a
magnetic particle or a fiber optic sensor, such as those disclosed, for
example, in U.S.
Patent No. 5,359,681. The binding agent may be immobilized on the solid
support
using a variety of techniques known to those of skill in the art, which are
amply
described in the patent and scientific literature. In the context of the
present invention,
the term "immobilization" refers to both noncovalent association, such as
adsorption,
and covalent attachment (which may be a direct linkage between the agent and
functional groups on the support or may be a linkage by way of a cross-linking
agent).
Immobilization by adsorption to a well in a microtiter plate or to a membrane
is
2o preferred. In such cases, adsorption may be achieved by contacting the
binding agent,
in a suitable buffer, with the solid support for a suitable amount of time.
The contact
time varies with temperature, but is typically between about 1 hour and about
1 day. In
general, contacting a well of a plastic microtiter plate (such as polystyrene
or
polyvinylchloride) with an amount of binding agent ranging from about 10 ng to
about
10 fig, and preferably about 100 ng to about 1 fig, is sufficient to
immobilize an
adequate amount of binding agent.
Covalent attachment of binding agent to a solid support may generally be
achieved by first reacting the support with a bifunctional reagent that will
react with
both the support and a functional group, such as a hydroxyl or amino group, on
the
3o binding agent. For example, the binding agent may be covalently attached to
supports
having an appropriate polymer coating using benzoquinone or by condensation of
an
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aldehyde group on the support with an amine and an active hydrogen on the
binding
partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at
A12-A13).
In certain embodiments, the assay is a two-antibody sandwich assay.
This assay may be performed by first contacting an antibody that has been
immobilized
on a solid support, commonly the well of a microtiter plate, with the sample,
such that
polypeptides within the sample are allowed to bind to the immobilized
antibody.
Unbound sample is then removed from the immobilized polypeptide-antibody
complexes and a detection reagent (preferably a second antibody capable of
binding to a
l0 different site on the polypeptide) containing a reporter group is added.
The amount of
detection reagent that remains bound to the solid support is then determined
using a
method appropriate for the specific reporter group.
More specifically, once the antibody is immobilized on the support as
described above, the remaining protein binding sites on the support are
typically
~ 5 blocked. Any suitable blocking agent known to those of ordinary skill in
the art, such
as bovine serum albumin or Tween 20TM (Sigma Chemical Co., St. Louis, MO). The
immobilized antibody is then incubated with the sample, and polypeptide is
allowed to
bind to the antibody. The sample may be diluted with a suitable diluent, such
as
phosphate-buffered saline (PBS) prior to incubation. In general, an
appropriate contact
2o time (i.e., incubation time) is a period of time that is sufficient to
detect the presence of
polypeptide within a sample obtained from an individual with ovarian cancer.
Preferably, the contact time is sufficient to achieve a level of binding that
is at least
about 95% of that achieved at equilibrium between bound and unbound
polypeptide.
Those of ordinary skill in the art will recognize that the time necessary to
achieve
25 equilibrium may be readily determined by assaying the level of binding that
occurs over
a period of time. At room temperature, an incubation time of about 30 minutes
is
generally sufficient.
Unbound sample may then be removed by washing the solid support
with an appropriate buffer, such as PBS containing 0.1% Tween 20TM. The second
3o antibody, which contains a reporter group, may then be added to the solid
support.
Preferred reporter groups include those groups recited above.
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The detection reagent is then incubated with the immobilized antibody-
polypeptide complex for an amount of time sufficient to detect the bound
polypeptide.
An appropriate amount of time may generally be determined by assaying the
level of
binding that occurs over a period of time. Unbound detection reagent is then
removed
and bound detection reagent is detected using the reporter group. The method
employed for detecting the reporter group depends upon the nature of the
reporter
group. For radioactive groups, scintillation counting or autoradiographic
methods are
generally appropriate. Spectroscopic methods may be used to detect dyes,
luminescent
groups and fluorescent groups. Biotin may be detected using avidin, coupled to
a
t o different reporter group (commonly a radioactive or fluorescent group or
an enzyme).
Enzyme reporter groups may generally be detected by the addition of substrate
(generally for a specific period of time), followed by spectroscopic or other
analysis of
the reaction products.
To determine the presence or absence of a cancer, such as ovarian
cancer, the signal detected from the reporter group that remains bound to the
solid
support is generally compared to a signal that corresponds to a predetermined
cut-off
value. In one preferred embodiment, the cut-off value for the detection of a
cancer is
the average mean signal obtained when the immobilized antibody is incubated
with
samples from patients without the cancer. In general, a sample generating a
signal that
2o is three standard deviations above the predetermined cut-off value is
considered positive
for the cancer. In an alternate preferred embodiment, the cut-off value is
determined
using a Receiver Operator Curve, according to the method of Sackett et al.,
Clinical
Epidemiolo~: A Basic Science for Clinical Medicine, Little Brown and Co.,
1985,
p. 106-7. Briefly, in this embodiment, the cut-off value may be determined
from a plot
of pairs of true positive rates (i.e., sensitivity) and false positive rates
(100%-specificity)
that correspond to each possible cut-off value for the diagnostic test result.
The cut-off
value on the plot that is the closest to the upper left-hand corner (i.e., the
value that
encloses the largest area) is the most accurate cut-off value, and a sample
generating a
signal that is higher than the cut-off value determined by this method may be
considered
3o positive. Alternatively, the cut-off value may be shifted to the left along
the plot, to
minimize the false positive rate, or to the right, to minimize the false
negative rate. In
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general, a sample generating a signal that is higher than the cut-off value
determined by
this method is considered positive for a cancer.
In a related embodiment, the assay is performed in a flow-through or
strip test format, wherein the binding agent is immobilized on a membrane,
such as
nitrocellulose. In the flow-through test, polypeptides within the sample bind
to the
immobilized binding agent as the sample passes through the membrane. A second,
labeled binding agent then binds to the binding agent-polypeptide complex as a
solutiori
containing the second binding agent flows through the membrane. The detection
of
bound second binding agent may then be performed as described above. In the
strip test
to format, one end of the membrane to which binding agent is bound is immersed
in a
solution containing the sample. The sample migrates along the membrane through
a
region containing second binding agent and to the area of immobilized binding
agent.
Concentration of second binding agent at the area of immobilized antibody
indicates the
presence of a cancer. Typically, the concentration of second binding agent at
that site
generates a pattern, such as a line, that can be read visually. The absence of
such a
pattern indicates a negative result. In general, the amount of binding agent
immobilized
on the membrane is selected to generate a visually discernible pattern when
the
biological sample contains a level of polypeptide that would be sufficient to
generate a
positive signal in the two-antibody sandwich assay, in the format discussed
above.
Preferred binding agents for use in such assays are antibodies and antigen-
binding
fragments thereof. Preferably, the amount of antibody immobilized on the
membrane
ranges from about 25 ng to about lpg, and more preferably from about 50 ng to
about
500 ng. Such tests can typically be performed with a very small amount of
biological
sample.
Of course, numerous other assay protocols exist that are suitable for use
with the tumor proteins or binding agents of the present invention. The above
descriptions are intended to be exemplary only. For example, it will be
apparent to
those of ordinary skill in the art that the above protocols may be readily
modified to use
tumor polypeptides to detect antibodies that bind to such polypeptides in a
biological
3o sample. The detection of such tumor protein specific antibodies may
correlate with the
presence of a cancer.
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A cancer may also, or alternatively, be detected based on the presence of
T cells that specifically react with a tumor protein in a biological sample.
Within
certain methods, a biological sample comprising CD4+ and/or CD8+ T cells
isolated
from a patient is incubated with a tumor polypeptide, a polynucleotide
encoding such a
polypeptide and/or an APC that expresses at least an immunogenic portion of
such a
polypeptide, and the presence or absence of specific activation of the T cells
is detected.
Suitable biological samples include, but are not limited to, isolated T cells.
For
example, T cells may be isolated from a patient by routine techniques (such as
by
Ficoll/Hypaque density gradient centrifugation of peripheral blood
lymphocytes). T
1o cells may be incubated in vitro for 2-9 days (typically 4 days) at
37°C with polypeptide
(e.g., 5 - 25 p,g/ml). It may be desirable to incubate another aliquot of a T
cell sample in
the absence of ovarian tumor polypeptide to serve as a control. For CD4+ T
cells,
activation is preferably detected by evaluating proliferation of the T cells.
For CD8+ T
cells, activation is preferably detected by evaluating cytolytic activity. A
level of
proliferation that is at least two fold greater and/or a level of cytolytic
activity that is at
least 20% greater than in disease-free patients indicates the presence of a
cancer in the
patient.
As noted above, a cancer may also, or alternatively, be detected based on
the level of mRNA encoding a ovarian tumor protein in a biological sample. For
example, at least two oligonucleotide primers may be employed in a polymerase
chain
reaction (PCR) based assay to amplify a portion of a tumor cDNA derived from a
biological sample, wherein at least one of the oligonucleotide primers is
specific for
(i. e., hybridizes to) a polynucleotide encoding the tumor protein. The
amplified cDNA
is then separated and detected using techniques well known in the art, such as
gel
electrophoresis. Similarly, oligonucleotide probes that specifically hybridize
to a
polynucleotide encoding a tumor protein may be used in a hybridization assay
to detect
the presence of polynucleotide encoding the tumor protein in a biological
sample.
To permit hybridization under assay conditions, oligonucleotide primers
and probes should comprise an oligonucleotide sequence that has at least about
60%,
3o preferably at least about 75% and more preferably at least about 90%,
identity to a
portion of a polynucleotide encoding a tumor protein of the invention that is
at least 10
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nucleotides, and preferably at least 20 nucleotides, in length. Preferably,
oligonucleotide primers and/or probes hybridize to a polynucleotide encoding a
polypeptide described herein under moderately stringent conditions, as defined
above.
Oligonucleotide primers and/or probes which may be usefully employed in the
diagnostic methods described herein preferably are at least 10-40 nucleotides
in length.
In a preferred embodiment, the oligonucleotide primers comprise at least 10
contiguous
nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA
molecule
having a sequence as disclosed herein. Techniques for both PCR based assays
and
hybridization assays are well known in the art (see, for example, Mullis et
al., Cold
to Spring Harbor Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology,
Stockton
Press, NY, 1989).
One preferred assay employs RT-PCR, in which PCR is applied in
conjunction with reverse transcription. Typically, RNA is extracted from a
biological
sample, such as biopsy tissue, and is reverse transcribed to produce cDNA
molecules.
PCR amplification using at least one specific primer generates a cDNA
molecule, which
may be separated and visualized using, for example, gel electrophoresis.
Amplification
may be performed on biological samples taken from a test patient and from an
individual who is not afflicted with a cancer. The amplification reaction may
be
performed on several dilutions of cDNA spanning two orders of magnitude. A two-
fold
or greater increase in expression in several dilutions of the test patient
sample as
compared to the same dilutions of the non-cancerous sample is typically
considered
positive.
In another embodiment, the compositions described herein may be used
as markers for the progression of cancer. In this embodiment, assays as
described
above for the diagnosis of a cancer may be performed over time, and the change
in the
level of reactive polypeptide(s) or polynucleotide(s) evaluated. For example,
the assays
may be performed every 24-72 hours for a period of 6 months to 1 year, and
thereafter
performed as needed. In general, a cancer is progressing in those patients in
whom the
level of polypeptide or polynucleotide detected increases over time. In
contrast, the
3o cancer is not progressing when the level of reactive polypeptide or
polynucleotide either
remains constant or decreases with time.
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Certain in vivo diagnostic assays may be performed directly on a tumor.
One such assay involves contacting tumor cells with a binding agent. The bound
binding agent may then be detected directly or indirectly via a reporter
group. Such
binding agents may also be used in histological applications. Alternatively,
polynucleotide probes may be used within such applications.
As noted above, to improve sensitivity, multiple tumor protein markers
may be assayed within a given sample. It will be apparent that binding agents
specific
for different proteins provided herein may be combined within a single assay.
Further,
multiple primers or probes may be used concurrently. The selection of tumor
protein
t o markers may be based on routine experiments to determine combinations that
results in
optimal sensitivity. In addition, or alternatively, assays for tumor proteins
provided
herein may be combined with assays for other known tumor antigens.
The present invention further provides kits for use within any of the
above diagnostic methods. Such kits typically comprise two or more components
necessary for performing a diagnostic assay. Components may be compounds,
reagents, containers and/or equipment. For example, one container within a kit
may
contain a monoclonal antibody or fragment thereof that specifically binds to a
tumor
protein. Such antibodies or fragments may be provided attached to a support
material,
as described above. One or more additional containers may enclose elements,
such as
2o reagents or buffers, to be used in the assay. Such kits may also, or
alternatively, contain
a detection reagent as described above that contains a reporter group suitable
for direct
or indirect detection of antibody binding.
Alternatively, a kit may be designed to detect the level of mRNA
encoding a tumor protein in a biological sample. Such kits generally comprise
at least
one oligonucleotide probe or primer, as described above, that hybridizes to a
polynucleotide encoding a tumor protein. Such an oligonucleotide may be used,
for
example, within a PCR or hybridization assay. Additional components that may
be
present within such kits include a second oligonucleotide and/or a diagnostic
reagent or
container to facilitate the detection of a polynucleotide encoding a tumor
protein.
3o The following Examples are offered by way of illustration and not by
way of limitation.
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EXAMPLES
Example 1
Identification of Representative Ovarian Carcinoma cDNA Sequences
This Example illustrates the identification of ovarian tumor cDNA
molecules.
Primary ovarian tumor and metastatic ovarian tumor cDNA libraries
were each constructed in kanamycin resistant pZErOT""-2 vector (Invitrogen)
from pools
to of three different ovarian tumor RNA samples. For the primary ovarian tumor
library,
the following RNA samples were used: (1) a moderately differentiated papillary
serous
carcinoma of a 41 year old, (2) a stage IIIC ovarian tumor and (3) a papillary
serous
adenocarcinoma for a 50 year old Caucasian. For the metastatic ovarian tumor
library,
the RNA samples used were omentum tissue from: (1) a metastatic poorly
differentiated papillary adenocarcinoma with psammoma bodies in a 73 year old,
(2) a
metastatic poorly differentiated adenocarcinoma in a 74 year old and (3) a
metastatic
poorly differentiated papillary adenocarcinoma in a 68 year old.
The number of clones in each library was estimated by plating serial
dilutions of unamplified libraries. Insert data were determined from 32
primary ovarian
2o tumor clones and 32 metastatic ovarian tumor clones. The library
characterization
results are shown in Table I.
Table I
Characterization of cDNA Libraries
# ClonesClones Insert Ave. Insert
with Size
Library in LibraryInsert Range (bp)Size (bp)
(%)
Primary Ovarian 1,258,00097 175 - 80002356
Tumor
Metastatic Ovarian 1,788,000100 150 - 43001755
Tumor
Four subtraction libraries were constructed in ampicillin resistant
pcDNA3.1 vector (Invitrogen). Two of the libraries were from primary ovarian
tumors
and two were from metastatic ovarian tumors. In each case, the number of
restriction
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enzyme cuts within inserts was minimized to generate full length subtraction
libraries.
The subtractions were each done with slightly different protocols, as
described in more
detail below.
A. POTS 2 Library: Primary Ovarian Tumor Subtraction Library
Tracer: 10 Pg primary ovarian tumor library, digested with Not I
Driver: 35 ~g normal pancreas in pcDNA3.1(+)
20 Pg normal PBMC in pcDNA3.1 (+)
~g normal skin in pcDNA3.1 (+)
10 35 ~g normal bone marrow in pZErOT""-2
Digested with Bam HI/Xho I/Sca I
Two hybridizations were performed, and Not I-cut pcDNA3.1 (+) was the
cloning vector for the subtracted library. Sequence results for previously
unidentified
clones that were randomly picked from the subtracted library are presented in
Table II.
Table II
Ovarian Carcinoma Seauences
Sequence SEQ ID NO
21909 2
21920 9
21921 10
25099 143
25101 144
25103 145
25107 146
25111 148
25113 149
25115 150
25116 151
25752 156
25757 158
25769 161
21907 1
21911
25763 160
25770 162
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B. POTS 7 Library: Primary Ovarian Tumor Subtraction Libr
Tracer: 10 ~g primary ovarian tumor library, digested with Not I
Driver: 35 ~g normal pancreas in pcDNA3.1 (+)
20 pg normal PBMC in pcDNA3.1 (+)
10 pg normal skin in pcDNA3.1(+)
35 pg normal bone marrow in pZErOT""-2
Digested with Bam HI/Xho I/Sca I
~25 p,g pZErOT""-2, digested with Bam HI and Xho I
Two hybridizations were performed, and Not I-cut pcDNA3.1 (+) was the
l0 cloning vector for the subtracted library. Sequence results for previously
unidentified
clones that were randomly picked from the subtracted library are presented in
Table III.
Table III
Ovarian Carcinoma Sequences
Sequence SEQ ID NO
24937 125
24940 128
24946 132
24950 133
24951 134
24956 137
25791 166
25796 167
25797 168
25804 171
24955 136
C. OS1D Library: Metastatic Ovarian Tumor Subtraction Library
Tracer: 10~g metastatic ovarian library in pZErOT""-2, digested
2o with Not I
Driver: 24.S~g normal pancreas in pcDNA3.1
14~g normal PBMC in pcDNA3.1
l4p,g normal skin in pcDNA3.1
24.Spg normal bone marrow in pZErOT""-2
SOp,g pZErOT""-2, digested with Bam HI/Xho I/Sfu I
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Three hybridizations were performed, and the last two hybridizations
were done with an additional l5pg of biotinylated pZErOT""-2 to remove
contaminating
pZErOT""-2 vectors. The cloning vector for the subtracted library was
pcDNA3.1/Not I
cut. Sequence results for previously unidentified clones that were randomly
picked
from the subtracted library are presented in Table IV.
Table IV
Ovarian Carcinoma Sequences
Sequence SEQ ID NO
24635 57
24647 63
24661 69
24663 70
24664 71
24670 72
24675 75
23645.1 13
23660.1 16
23666.1 19
23679.1 23
24651 65
24683 78
to
D. OS1F Library: Metastatic Ovarian Tumor Subtraction Library
Tracer: l Opg metastatic ovarian tumor library, digested with Not
I
Driver: 12.8~g normal pancreas in pcDNA3.1
7.3pg normal PBMC in pcDNA3.1
7.3p.g normal skin in pcDNA3.1
12.8~g normal bone marrow in pZErOT""-2
25~g pZErOT""-2, digested with Bam HI/Xho I/Sfu I
One hybridization was performed. The cloning vector for the subtracted
library was pcDNA3.1/Not I cut. Sequence results for previously unidentified
clones
that were randomly picked from the subtracted library are presented in Table
V.
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Table V
Ovarian Carcinoma Sequences
Sequence SEQ ID NO
24344 33
24356 42
24368 53
24696 86
24699 89
24701 90
24703 91
24707 95
24709 97
24732 111
24745 120
24746 121
24337 28
24348 35
24351 38
24358 44
24360 46
24361 47
24690 81
24692 82
24694 84
24705 93
24711 98
24713 99
24727 107
24741 117
24359 (78% Human mRNA for KIAA011145
gene, complete cds)
24336 (79% with H. sapiens mitochondria)27
genome (consensus sequence))
24737 (84%Human ADP/ATP translocase114
mRNA)
24363 (87% Homo Sapiens eukaryotic49
translation elongation factor
1 alpha 1 (EEF 1 A 1 )
24357 (87% S. scrofa mRNA for 43
UDP glucose
pyrophosphorylase)
24362 (88% Homo Sapiens Chromosome48
16
BAC clone CIT987SK-A-233A7)
24704 (88% Homo Sapiens chromosome92
9, clone
hRPK.401_G_18)
24367 (89% Homo Sapiens 12p13.3 52
BAC
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Sequence SEQ ID NO
RCPI l 1-935C2)
24717 (89% Homo Sapiens proliferation-103
associated gene A (natural killer-enhancing
factor A) (PAGA)
24364 (89%Human DNA sequence from50
PAC
27K14 on chromosome Xp11.3-Xp11.4)
24355 (91% Homo sapiens chromosome41
17,
clone hCIT.91_J_4)
24341 (91 %Homo Sapiens chromosome32
5, BAC
clone 249h5 (LBNL H 149)
24714 (91 %Human DNA sequence 100
from clone
125N5 on chromosome 6q26-27)
The sequences in Table VI, which correspond to known sequences, were
also identified in the above libraries.
Table VI
Ovarian Carcinoma Sequences
Identity SEQ ID SequenceLibrar
NO Y
Genomic sequence from Human 9q34 56 24634 OS1D
Homo Sapiens 12p13.3 PAC RPCII-96H9 66 24653 OS1D
(Roswell
Park Cancer Institute Human PACLibrary)
Homo Sapiens annexin II (lipocortin 60 24640 OS
II) (ANX2) 1
mRNA D
Homo sapiens eukaryotic translation 55 24627 OS1D
elongation factor
1 alpha 1 (EEF 1 A 1 )
Homo Sapiens ferritin, heavy polypeptide64 24648 OS1D
1 (FTHI)
Homo Sapiens FK506-binding protein 22 23677.1 OS
1 A ( 12kD) 1
(FKBP 1 A) mRNA D
Homo Sapiens growth arrest specific 73 24671 OS
transcript 5 gene 1
D
Homo Sapiens keratin 18 (KRT18) mRNA 68 24657 OS1D
Homo sapiens mRNA; cDNA DKFZp564H182 76 24677 OS1D
Homo Sapiens ribosomal protein S7 74 24673 OS1D
(RPS7)
Homo Sapiens ribosomal protein, large,14 23647.1 OS1D
PO (RPLPO)
mRNA
Homo Sapiens T cell-specific tyrosine67 24655 OS1D
kinase mRNA
Homo Sapiens tubulin, alpha, ubiquitous61 24642 OS
(K-ALPHA- 1
1) D
HSU78095 Homo Sapiens placental bikunin18 23662.1 OS1D
mRNA
Human BAC clone GSOSSK18 from 7p15-p2111 23636.1 OS1D
91
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Identity SEQ ID SequenceLibrar
NO Y
Human insulin-like growth factor-binding58 24636 OS1D
protein-3
gene
Human mRNA for ribosomal protein 79 24687 OS
1
D
Human non-histone chromosomal protein62 24645 OS1D
HMG-14
mRNA
Human ribosomal protein L3 mRNA, 3' 59 24638 OS1D
end
Human TSC-22 protein mRNA 77 24679 OS1D
HUMGFIBPA Human growth hormone-dependent12 23637.1 OS1D
insulin-like growth factor-binding
protein
HUMMTA Homo Sapiens mitochondrial 17 23661.1 OS1D
DNA
~HUMMTCG Human mitochondrion 21 23673.1 OS1D
HUMTI227HC Human mRNA for TI-227H 20 23669.1 OS
1
D
HUMTRPM2A Human TRPM-2 mRNA 15 23657.1 OS1D
Genomic sequence from Human 13 80 24689 OS
1
F
H.sapiens CpG island DNA genomic Msel104 24719 OS1F
fragment,
clone 84a5
H.sapiens RNA for snRNP protein B 110 24730 OS1F
Homo Sapiens (clone L6) E-cadherin 108 24728 OS
(CDH 1 ) gene 1
F
Homo Sapiens atrophin-1 interacting 37 24350 OS1F
protein 4 (AIP4)
mRNA
Homo Sapiens CGI-08 protein mRNA 102 24716 OS1F
Homo Sapiens clone 24452 mRNA sequence54 24374 OS1F
Homo Sapiens clone IMAGE 286356 83 24693 OS
1
F
Homo sapiens cornichon protein mRNA 113 24735 OS1F
Homo Sapiens hypothetical 43.2 Kd 87 24697 OS
protein mRNA 1
F
Homo Sapiens interleukin 1 receptor 29 24338 OS1F
accessory protein
(IL 1 RAP) mRNA.
Homo Sapiens K-Cl cotransporter KCC4 31 24340 OS1F
mRNA,
complete cds
Homo sapiens keratin 8 (KRTB) mRNA 11 S 24739 OS
1F
Homo Sapiens mRNA for DEPP (decidual 36 24349 OS1F
protein
induced by progesterone)
Homo Sapiens mRNA for KIAA0287 gene 1 O 1 24715 OS
1
F
Homo sapiens mRNA for KIAA0762 protein118 24742 OS
1
F
Homo Sapiens mRNA for zinc-finger 24 24333 OS1F
DNA-binding
protein, complete cds
Homo Sapiens mRNA; cDNA DKFZp434K114 112 24734 OS1F
Homo Sapiens mRNA; cDNA DKFZp564E196225 24334 OS1F
(from
clone DKFZp564E1962)
Homo Sapiens nuclear chloride ion 34 24345 OS1F
channel protein
(NCC27) mRNA
Homo Sapiens ribosomal protein L13 109 24729 OS1F
(RPL13)
Homo Sapiens senescence-associated 94 24706 OS
epithelial 1
F
92
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Identity SEQ ID SequenceLibrar
NO
y
membrane protein (SEMP1)
Homo Sapiens tumor protein, translationally-26 24335 OS1F
controlled 1 (TPT 1 ) mRNA.
Homo Sapiens tumor suppressing subtransferable51 24366 OS1F
candidate 1 (TSSC1)
Homo Sapiens v-fos FBJ marine osteosarcoma85 24695 OS
viral 1
F
oncogene homolog(FOS) mRNA
Homo Sapiens zinc finger protein slug106 24722 OS1F
(SLUG) gene
Human clone 23722 mRNA 105 24721 OS1F
Human clones 23667 and 23775 zinc 119 24744 OS1F
finger protein
mRNA
Human collagenase type IV mRNA, 3' 39 24352 OS1F
end.
Human DNA sequence from PAC 29K1 on 116 24740 OS1F
chromosome 6p21.3-22.2.
Human ferritin H chain mRNA 96 24708 OS
1
F
Human heat shock protein 27 (HSPB 88 24698 OS
1 ) gene exons 1- 1
F
3
Human mRNA for KIAA0026 gene 30 24339 OS1F
Human mRNA for T-cell cyclophilin 40 24354 OS1F
Genomic sequence from Human 9q34, 140 25092 POTS2
complete
sequence [Homo Sapiens]
H.sapiens DNA for muscle nicotinic 3 21910 POTS2
acetylcholine
receptor gene promotor, clone ICRFc105F02104
Homo sapiens breast cancer suppressor142 25098 POTS2
candidate 1
(bcsc-1) mRNA, complete cds
Homo sapiens CGI-151 protein mRNA, 8 21916 POTS2
complete cds
Homo sapiens complement component 4 21913 POTS2
3 (C3) gene,
exons 1-30.
Homo Sapiens mRNA for hepatocyte growth159 25758 POTS2
factor
activator inhibitor type 2,complete
cds
Homo Sapiens preferentially expressed153 25745 POTS2
antigen of
melanoma (PRAME) mRNA
Homo Sapiens prepro dipeptidyl peptidase152 25117 POTS2
I (DPP-I)
gene, complete cds
Homo sapiens SKB 1 (S. cerevisiae) 147 25110 POTS2
homolog (SKB 1 )
mRNA.
Homo Sapiens SWI/SNF related, matrix 6 21914 POTS2
associated,
actin dependent regulator of chromatin,
subfamily a,
member 4 (SMARCA4)
Human 12S RNA induced by poly(rI), 155 25749 POTS2
poly(rC) and
Newcastle disease virus
Human ferritin Heavy subunit mRNA, 7 21915 POTS2
complete cds.
Human glyceraldehyde-3-phosphate dehydrogenase141 25093 POTS2
93
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Identity SEQ ID SequenceLibrar
NO Y
(GAPDH) mRNA, complete cds.
Human mRNA for fibronectin (FN precursor)157 25755 POTS2
Human translocated t(8;14) c-myc (MYC)154 25746 POTS2
oncogene,
exon 3 and complete cds
H.sapiens vegf gene, 3'UTR 169 25799 POTS?
Homo Sapiens 305 ribosomal protein 170 25802 POTS7
S7 homolog
mRNA, complete cds
Homo Sapiens acetyl-Coenzyme A acetyltransferase172 25808 POTS?
2
(acetoacetyl Coenzyme A thiolase)
(ACAT2) mRNA
Homo Sapiens amyloid beta precursor 138 24959 POTS?
protein-binding
protein 1, 59kD (APPBP1) mRNA.
Homo Sapiens arylacetamide deacetylase129 24942 POTS7
(esterase)
(AADAC) mRNA.
Homo Sapiens clone 23942 alpha enolase165 25787 POTS7
mRNA,
partial cds
Homo Sapiens echinoderm microtubule-associated130 24943 POTS?
protein-like EMAP2 mRNA, complete
cds
Homo Sapiens IMP (inosine monophosphate)164 25775 POTS?
dehydrogenase 2 (IMPDH2) mRNA
Homo Sapiens megakaryocyte potentiating126 24938 POTS?
factor
(MPF) mRNA.
Homo Sapiens mRNA for KIAA0552 protein,163 25771 POTS7
complete cds
Homo Sapiens Norrie disease protein 173 25809 POTS7
(NDP) mRNA
Homo Sapiens podocalyxin-like (PODXL)131 24944 POTS7
mRNA.
Homo Sapiens synaptogyrin 2 (SYNGR2) 135 24952 POTS7
mRNA.
Human aldose reductase mRNA, complete139 24969 POTS7
cds.
Human cyclooxygenase-1 (PTSG1) mRNA, 124 24935 POTS7
partial
cds
Human H19 RNA gene, complete cds. 122 24933 POTS?
Human mRNA for Apol Human (MERS(Aopl-127 24939 POTS?
Mouse)-like protein), complete cds
Human triosephosphate isomerase mRNA,123 24934 POTS7
complete
cds.
Still further ovarian carcinoma polynucleotide and/or polypeptide
sequences identified from the above libaries are provided below in Table VII.
Sequences 05745 (SEQ ID NOs: 183 & 185), 05845 (SEQ ID NO: 193) and 05855
(SEQ ID NO: 194) represent novel sequences. The remaining sequences exhibited
at
least some homology with known genomic and/or EST sequences.
94
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Table VII
SEQ ID: Sequence Library
174: 05655 CRABP OS1D
175 : 05665 Ceruloplasmin POTS2
176: 0567S 41191.SEQ(1>487) POTS2
177: 05685 KIAA0762.seq(1>3999)POTS7
178 : 05695 41220.seq(1>1069) POTS7
179: 05705 41215.seq(1>1817) POTS2
180: 05715 41213.seq(1>2382) POTS2
181 : 05725 41208.seq(1>2377) POTS2
182 : 0573S 41177.seq(1>1370) OS1F
183 : 05745 47807.seq(1>2060) n/a
184 : 05685/VSGF DNA seq n/a
185: 0574S 47807.seq(1>3000) n/a
186: 05685/VSGF protein seq n/a
187 : 449H1(57581) OS1D
188: 451 E 12(575 82) OS 1 D
189: 453C7 3'(57583.1)OsteonectinOS1D
190: 453C7 5'(57583.2) OS1D
191: 45661 3'(57584.1)NeurotensinOS1F
192: 45661 5'(57584.2) OS1F
193: 0584S 46565(57585) OS1F
194: 05855 469B12(57586) POTS2
195: 05695 474C3(57587) POTS7
196: 483B1_3'(24934.1)TriosephosphatePOTS?
197: 57885 Human preferentiallyPOTS2
expressed antigen of melanoma
198: 57886 Chromosome 22q12.1 POTS2
clone
CTA-723E4
199: 57887 Homologous to mousePOTS2
brain
cDNA clone MNCb-0671
From the foregoing it will be appreciated that, although specific
embodiments of the invention have been described herein for purposes of
illustration,
various modifications may be made without deviating from the spirit and scope
of the
invention. Accordingly, the invention is not limited except as by the appended
claims.
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SEQUENCE LISTING
<110> Corixa Corporation
Xu, Jiangchun
Stolk, John A.
<120> OVARIAN TUMOR SEQUENCES AND
METHODS OF USE THEREFOR
<130> 210121.484PC
<140> PCT
<141> 2000-09-08
<160> 199
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
1
caacctcactagtaaatgaaagaaatattgtaatttgtatttgatctgctgggtctttgg 60
agtcagaactggttttatcagcagtttgatcttctgaggtctggtatgtagtttgctggc 120
ccacagaaccttcacgtgtattcacagcctcaatgccataaggaaactcttttagaagtt 180
ctgacagctggtcatgtaggtataagacaggtgccttatcactgtggatttcatttcttg 240
caggatcttggggagtatagttgctggatgcatctatttcctgagggtaaatatcctcct 300
ggncgacgcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactg 360
tgccttctanttgccanccatntgttgtttgcccct 396
<210> 2
<211> 396
<212> DNA
<213> Homo sapien
<400>
2
cgaccaaaaagtaaactccaagtgaacatcaaatcaaatctaatccttttggccacatga 60
ctggttgttctttatctcatagttacaatgaatcatataaactgtagactgccactacca 120
cgatacttctgtgacacagaaggaatgtcctatttgcctatctatctgaggaatgttaaa 180
tagagaaaaatagattataaaacaacctggaggtcacaggattctgagataatccctctg 240
ttaaaaaacatctgaacagcaaatgtccaatctgtaataaaatagttaaaggtccaagtc 300
aagtccacttctacttggctggcccagcacaagaaatctaacagcactttgtaatcattt 360
tgcttttctaattttcccggaggacatgggccattg 396
<210> 3
<211> 396
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2
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
3
cgcccttttttttttttttttnattggnnnaantcnctttnantnnaaaaacntgnangg 60
naancccanncccnnggnaccannnccaggagttgggtgganactgagtggggtttgtgt 120
gggtgagggggcatctactcctnttgcaacaagccaaaagtagaacagcctaaggaaaag 180
tgacctgccttggagccttagtccctcccttagggccccctcagcctaccctatccaagt 240
ctgaggctatggaagtctccctcctagttcactagcaggttccccatcttttccaggctg 300
cccctagcactccacgtttttctgaaaaaatctanacaggccctttttgggtacctaaaa 360
cccagctgaggttgtgagcttgtaaggtaaagcaag 396
<210> 4
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 4
gaccaatccttgncncactancaaaangaccccnctnaccnccaggaactgaacctnnnt 60
gtnnacctccnnctgcnnagccntatntccaanatcacccaccgtatccactgggaatct 120
gccagcctcctgcgatcagaagagaccaatcgaaaatgagggtttcacantcacagctga 180
aggaaaaggccaaggcaccttgtcggnggngacaatgtaccatgctaaggccaaagatca 240
actcacctgtaataaattcgacctcaaggtcaccataaaaccagcaccggaacagaaaaa 300
gaggcctnaggatgcccaagaaacacttttgatcctttgaaaactgtaccaaggtaccgg 360
ggggagacccaggaaaggnccnttatgtntnnntnt 396
<210> 5
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 5
gacgccggagctgccgcgccagtcgcctagcaggtcctctaccggcttattcctgtgccg 60
gatcttcatcggcacaggggccactgagacgtttctgcctccctctttcttcctccgctc 120
tttctcttccctctngtttagtttgcctgggagcttgaaaggagaaagcacnggggtcgc 180
cccaaaccctttctgcttctgcccatcacaagtgccactaccgccatgggcctcactatc 240
tcctccctcttctcccgactatttggcaagaagcagatgcgcattttgatggttggattg 300
gatgctgctggcaagacaaccattcttgataaactgaaagtanggganataagnaccacc 360
atttctaccattgggtttaatgggggaaacagtana 396
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3
<210> 6
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 6
acgggaggcgccgggaagtcgacggcgccggcggctcctgcaggaggccactgtctgcag 60
ctcccgtgaagatgtccactccagacccacccctgggcggaactcctcggccaggtcctt 120
ccccgggccctgcccttcccctggagccatgctgggccctagcccgggtccctcgccggg 180
ctccgcccacagcatgatggggcccagcccangggccgccctcagcaggacaccccatcc 240
ccacccaggggcctggagggtaccctcaggacaacatgcaccagatgcacaagcccatgg 300
agtccatgcatgagaagggcatgtcggacgacccgcgctacaaccagatgaaaggaatgg 360
ggatgcggtcagggggccatgctgggatggggcccc 396
<210> 7
<211> 396
<212> DNA
<213> Homo sapien
<400> 7
acccgagagtcgtcggggtttcctgcttcaacagtgcttggacggaacccggcgctcgtt 60
ccccaccccggccggccgcccatagccagccctccgtcacctcttcaccgcaccctcgga 120
ctgccccaaggcccccgccgccgctccagcgccgcgcagccaccgccgccgccgccgcct 180
ctccttagtcgccgccatgacgaccgcgtccacctcgcaggtgcgccagaactaccacca 240
ggactcagaggccgccatcaaccgccagatcaacctggagctctacgcctcctacgttta 300
cctgtccatgtcttactactttgaccgcgatgatgtggctttgaagaactttgccaaata 360
ctttcttcaccaatctcatgaggagagggaacatgc 396
<210> 8
<211> 396
<212> DNA
<213> Homo sapien
<400>
8
cgacaacaaggttaataccttagttcttaacattttttttctttatgtgtagtgttttca 60
tgctaccttggtaggaaacttatttacaaaccatattaaaaggctaatttaaatataaat 120
aatataaagtgctctgaataaagcagaaatatattacagttcattccacagaaagcatcc 180
aaaccacccaaatgaccaaggcatatatagtatttggaggaatcaggggtttggaaggag 240
tagggaggagaatgaaggaaaatgcaaccagcatgattatagtgtgttcatttagataaa 300
agtagaaggcacaggagaggtagcaaaggccaggcttttctttggttttcttcaaacata 360
ggtgaaaaaaacactgccattcacaagtcaaggaac 396
<210> 9
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
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4
<223> n = A,T,C or G
<400> 9
tcgacatcgcggcaactttttgcggattgttcttgcttccaggctttgcgctgcaaatcc 60
agtgctaccagtgtgaagaattccagctgaacaacgactgctcctcccccgagttcattg 120
tgaattgcacggtgaacgttcaagacatgtgtcagaaagaagtgatggagcaaagtgccg 180
ggatcatgtaccgcaagtcctgtgcatcatcagcggcctgtctcatcgcctctgccgggt 240
accagtccttctgctccccagggaaactgaactcagtttgcatcagctgctgcaacaccc 300
ctctttgtaacgggccaaggnccaaaaaaaggggaaagttctgncctcggccctcaggcc 360
agggctccgcaccaccatcctgttcctcaaattagc 396
<210> 10
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 10
cctttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 60
ttttttttttttttttttttttttttttttttttttttttttttaaaaaaaaaannnttt 120
tttttttttnaaaaaaangggnnnnnttttttncccnnnngggnggggggggggnnnnnt 180
ttnaaanaaaaaaaccnnaaannnnnggggnnnannnaannncccnccccnaancnntaa 240
aaaannnggnaaaanagggggggnannnnnnnggggggnaaaanttttttttttttnaag 300
ggnnnggnaaaaaantnnnnnnntttttttttnnaanngggnnaaaaaaaaaaaaaaaaa 360
attttttngggntnaggggnngggggaaaancccna 396
<210> 11
<211> 396
<212> DNA
<213> Homo sapien
<400>
11
agaacacaggtgtcgtgaaaactacccctaaaagccaaaatgggaaaggaaaagactcat 60
atcaacattgtcgtcattggacacgtagattcgggcaagtccaccactactggccatctg 120
atctataaatgcggtggcatcgacaaaagaaccattgaaaaatttgagaaggaggctgct 180
gagatgggaaagggctccttcaagtatgcctgggtcttggataaactgaaagctgagcgt 240
gaacgtggtatcaccattgatatctccttgtggaaatttgagaccagcaagtactatgtg 300
actatcattgatgccccaggacacagagactttatcaaaaacatgattacagggacatct 360
caggctgactgtgctgtcctgattgttgctgctggt 396
<210> 12
<211> 396
<212> DNA
<213> Homo sapien
<400>
12
cgaaaacctttaaaccccggtcatccggacatcccaacgcatgctcctggagctcacagc 60
cttctgtggtgtcatttctgaaacaagggcgtggatccctcaaccaagaagaatgtttat 120
gtcttcaagtgacctgtactgcttggggactattggagaaaataaggtggagtcctactt 180
gtttaaaaaatatgtatctaagaatgttctagggcactctgggaacctataaaggcaggt 240
atttcgggccctcctcttcaggaatcttcctgaagacatggcccagtcgaaggcccagga 300
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tggcttttgc tgcggccccg tggggtagga gggacagaga gacagggaga gtcagcctcc 360
acattcagag gcatcacaag taatggcaca attctt 396
<210> 13
<211> 396
<212> DNA
<213> Homo sapien
<400>
13
accacaggctggccacaagaagcgctggagtgtgctggcggctgcaggcctacggggcct 60
ggtccggctgctgcacgtgcgtgccggcttctgctgcggggtcatccgagcccacaagaa 120
ggccatcgccaccctgtgcttcagccccgcccacgagacccatctcttcacggcctccta 180
tgacaagcggatcatcctctgggacatcggggtgcccaaccaggactacgaattccaggc 240
cagccagctgctcacactggacaccacctctatccccctgcgcctctgccctgtcgcctc 300
ctgcccggacgcccgcctgctggccggctgcgagggcggctgctgctgctgggacgtgcg 360
gctggaccagccccaaaagaggagggtgtgtgaagt 396
<210> 14
<211> 396
<212> DNA
<213> Homo sapien
<400>
14
acggcgtcctcgtggaagtgacatcgtctttaaaccctgcgtggcaatccctgacgcacc 60
gccgtgatgcccagggaagacagggcgacctggaagtccaactacttccttaagatcatc 120
caactattggatgattatccgaaatgtttcattgtgggagcagacaatgtgggctccaag 180
cagatgcagcagatccgcatgtcccttcgcgggaaggctgtggtgctgatgggcaagaac 240
accatgatgcgcaaggccatccgagggcacctggaaaacaacccagctctggagaaactg 300
ctgcctcatatccgggggaatgtgggctttgtgttcaccaaggaggacctcactgagatc 360
agggacatgttgctggccaataaggtgccagctgct 396
<210> 15
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 15
accgcgcgggcacagggtgccgctgaccgaggcgtgcaaagactccagaattggaggcat 60
gatgaagactctgctgctgtttgtggggctgctgctgacctgggagagtgggcaggtcct 120
gggggaccagacggtctcagacaatgagctccaggaaatgtccaatcagggaagtaagta 180
cgtcaataaggaaattcaaaatgcttgtcaacggggtgaaacagataaagactctcatag 240
aaaaaacaaacgaagagcgcaagacactgctcagcaacctagaagaagccaagaagaaga 300
aagaggatgccctaaatgagaccagggaatcanagacaaagctgaaggagctcccaggag 360
tgtgcaatgagaccatgatggccctctgggaagagt 396
<210> 16
<211> 396
<212> DNA
<213> Homo sapien
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
6
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 16
tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 60
tttttttttttttttttttttttttttttttttttttttttttttttttttttngggggg 120
nnnaaanttttttntnanannnnngggnaaaaaaaaaaaaaanaangggggnnntnnggc 180
ccnnnanaaaaaaanngnnaannaanccccccnnnnnnncccncnnntnnggaaananna 240
aaaccccccccngggnngggnnaaaaanncccnggggnantttttatnnnannccccccc 300
ccnggggggggnggaaaaaaaaaantncccccnannaaaannggggnccccccnttttnc 360
aaaangggggnccgggccccccnnantnttnggggg 396
<210> 17
<211> 396
<212> DNA
<213> Homo sapien
<400> 17
accacactaaccatataccaatgatggcgcgatgtaacacgagaaagcacataccaaggc 60
caccacacaccacctgtccaaaaaggccttcgatacgggataatcctatttattacctca 120
gaagtttttttcttcgcaggatttttctgagccttttaccactccagcctagcccctacc 180
ccccaactaggagggcactggcccccaacaggcatcaccccgctaaatcccctagaagtc 240
ccactcctaaacacatccgtattactcgcatcaggagtatcaatcacctgagctcaccat 300
agtctaatagaaaacaaccgaaaccaaataattcaagcactgcttattacaattttactg 360
ggtctctattttaccctcctacaagcctcagagtac 396
<210> 18
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
18
tttttttttttttttttttttttttttttttttttttttttttttttttantcnaaaggg 60
gaaggnccctttttattaaanttggncattttactttncttttttnaaaangctaanaaa 120
aaanttttntttntncttaaaaaaaccctnnatntcacnancaaaaaaaacnattcccnc 180
ntncnttttgtgataaaaaaaaaggcaatggaattcaacntancctaanaaaactttncc 240
tgggaggaaaaaaaattnntccgngggaaacacttggggctntccaaantgnanccatnc 300
tangaggaccntctntaagatttccaaangaaaccccttcctnccaaangnantaccccg 360
ntgcctacnncccataaaaaaaacctcanccntaan 396
<210> 19
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
7
<223> n = A,T,C or G
<400>
19
ttttttttttttttttttttttttttttttttttttttttttttttntggtctgggcttt 60
tattttacnaaaaanctaanggnaaanntncnttaaactaantngaanacaaagtnttaa 120
ngaaaaaggnctgggggnntcntttacaaaaanggncngggncanntttgggcttaaaan 180
ttcaaaaagggnncntcaaangggtttgcatttgcatgtttcancnctaaancgnangaa 240
naaacccnggngnccnctgggaaaagttnttnanctnccaaaanatnaantntttgnanc 300
agggnntttttgggnaaaaaaannanttccanaaactttccatcccctggntttgggttc 360
ggccttgngttttcggnatnatntccnttaangggg 396
<210> 20
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 20
ttttttttttttttttttttttttttctnaacaaaccctgttnttgggngggngngggta 60
taatactaagttganatgatntcatttacgggggaaggcnctttgtgaannaggccttat 120
ttctnttgncctttcgtacagggaggaatttgaagtaaananaaaccnacctggattact 180
ccggtctgaactcaaatcacgtaggactttaatcgttgaacaaacaaacctttaatagcg 240
gctgcnccattgggatgtcctgatccaacatcgaggncgtaaaccctattgttgatatgg 300
actctaaaaataggattgcgctgttatccctagggtaacttgttcccgtggtcaaagtta 360
ttggatcaattgagtataagtagttcgctttgactg 396
<210> 21
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 21
acatanatnttatactancattnaccatctcacttgnaggaanactantatatcnctcac 60
acctnatatcctncntactatgcctagaaggaataatactatngctgttnattatancta 120
ctntnataaccctnaacacccactccctcttanccaatattgtgcctattgccatactag 180
tntttgccgcctgcnaagcagnggngggcctanccntactagnctcaatctccaacacnt 240
atggcctanactacgtacataacctaaacctactcnaatgctaaaactaatcnncccaac 300
anttatnttactaccactgacatgactttccaaaaaacacatantttgaatcaacncanc 360
cacccacancctanttattancatcatcccIntact 396
<210> 22
<211> 396
<212> DNA
<213> Homo sapien
<220>
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
8
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
22
ttttttttttttttganaaaagccggcataaagcacttttattgcaataataaaacttga 60
gactcataaatggtgctgggggaagggtgcagcaacgatttctcaccaaatcactacaca 120
ggacagcaaaggggtgagaaggggctgagggaggaaaagccaggaaactgagatcagcag 180
agggagccaagcatcaaaaaacaggagatgctgaagctgcgatgaccagcatcattttct 240
taanagaacattcaaggatttgtcatgatggctgggctttcactgggtgttaagtctaca 300
aacagcaccttcaattgaaactgtcaattaaagttcttaagatttaggaagtggtggagc 360
ttggaaagttatgagattacaaaattcctgaaagtc 396
<210> 23
<211> 396
<212> DNA
<213> Homo sapien
<400> 23
acaaaggcggttccaagctaaggaattccatcagtgcttttttcgcagccaccaaattta 60
gcaggcctgtgaggttttcatatcctgaagagatgtattttaaagctttttttttttaat 120
gaaaaaatgtcagacacacacaaaagtagaatagtaccatggagtccccacgtacccagc 180
ctgcagcttcaacagttaccacatttgccaaccggagagactgccaaggcaggaaaaagc 240
cctggaaagcccacggcccctttttcccttgggtcagaggccttagagctggctgccaaa 300
gcagccaaccaaaggggcagctcagctccttcgtggcaccagcagtgttcctgatgcagt 360
tgaagagttgatgtctttgacaacatacggacactg 396
<210> 24
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 24
cgactatcctctcagattcttatctggcactaatttataactattatattatcagagact 60
atgtagcaatatatcagtgcacaggcgcatcccaggcctgtacagatgtatgtctacacg 120
taagtataaatgaatttgcataccaggttttacacttgcatctctaatagagattaaaaa 180
caacaaattggcctcttcctaagtatattaatatcatttatccttacattttatgcctcc 240
ccctaaattaatgactgagttggtggaaagcggctaggttttattcatactgttttttgt 300
tctcaacttcaanagtaatctacctctgaaaaatttntantttaatattnnnnnnnagga 360
atttgngccactttannncttncnntntnntnnccn 396
<210> 25
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
9
<400> 25
ttttttttttttttttttttgtcttttaaaaaatataaaagtgttattattttaaaacat 60
caagcattacagactgtaaaatcaattaanaactttctgtatatgaggacaaaaatacat 120
ttaanacatatacaanaagatgctttttcctgagtagaatgcaaacttttatattaagct 180
tctttgaattttcaaaatgtaaaataccaaggctttttcacatcagacaaaaatcaggaa 240
tgttcaccttcacatccaaaaagaaaaaaaaaaaaaanccaattttcaagttgaagttna 300
ncaanaatgatgtaaaatctgaaaaaagtggccaaaattttaanttncaacanannngnn 360
ncagntttnatggatctntnnnnnnncttcnnntnn 396
<210> 26
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
26
gacgctcccccctccccccgagcgccgctccggctgcaccgcgctcgctccgagtttcag 60
gctcgtgctaagctagcgccgtcgtcgtctcccttcagtcgccatcatgattatctaccg 120
ggacctcatcagccacgatgagatgttctccgacatctacaagatccgggagatcgcgga 180
cgggttgtgcctggaggtggaggggaagatggtcagtaggacagaaggtaacattgatga 240
ctcgctcattggtggaaatgcctccgctgaaggccccgagggcgaaggtacccgaaagca 300
cagtaatcactgnngncnatnttgtcatgaaccatcacctgcnngaaacaannttnacaa 360
aanaancctncnnnnannncctnnnnnattncnnnn 396
<210> 27
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1)...(396)
<223> n = A,T,C or G
<400> 27
tttttttttttttttttttttttttttttttttttttttttggctaaantttatgtatac 60
nggttnttcaaangngggggaggggggggggcatccatntanncncnccaggtttatggn 120
gggntnttntactattannanttttcncttcaaancnaaggnttntcaaatcatnaaaat 180
tattaanattncngctgntaaaaaaangaatgaaccnncnnanganagganntttcatgg 240
ggggnatgcatcggggnannccnaanaaccncggggccattcccganaggcccaaaaaat 300
gtttnnnnaaaaagggtaaanttacccccntnaantttatannnnaaannnnannnnagc 360
ccaannnttnnnnnnnnnnnnnnccnnnnannnnnn 396
<210> 28
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc feature
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
<222> (1)...(396)
<223> n = A,T,C or G
<400>
28
cgacctttttttttttttttatagatgaaagagggtttatttattaatatatgatagcct 60
tggctcaaaaaagacaaatgagggctcaaaaaggaattacagtaactttaaaaaatatat 120
taaacatatccaagatcctaaatatattattctccccaaaagctagctgcttccaaactt 180
gatttgatattttgcatgttttccctacgttgcttggtaaatatatttgcttctcctttc 240
tgcaatcgacgtctgacagctgatttttgctgttttgncaacntgacgtttcaccttntg 300
tttcaccanttctggaggaattgttnaacancttacancactgccttgaanaaannnnan 360
gcctcaaaagntcttgnnctatnctnnttcntnnnt 396
<210> 29
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 29
gacttgctcatttagagtttgcaggaggctccatactaggttcagtctgaaagaaatctc 60
ctaatggtgctatagagagggaggtaacagaaagactcttttagggcatttttctgactc 120
atgaaaagagcacagaaaaggatgtttggcaatttgtcttttaagtcttaaccttgctaa 180
tgtgaatactgggaaagtgatttttttctcactcgtttttgttgctccattgtaaagggc 240
ggaggtcagtcttagtggccttgagagttgcttttggcatttaaatattctaagagaatt 300
aactgtatttcctgtcacctattcactantgcangaaatatacttgctccaaataagtca 360
ntatgagaagtcactgtcaatgaaanttgntttgtt 396
<210> 30
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
30
tttttttttttttttttttgaaatttanaaacaaattttatttaagatctgaaatacaat 60
tcctaaaatatcaacttttccanaaaaccgtggctacacaataatgcattgcctctatca 120
tgttanaacgtgcattanactcaaatacaaaaaccatgaaacaaatcaccatccttcaac 180
aatttgagcaaagatagaatgcctaagaacaacatagatggacttgcagaggatgggctg 240
ttttacttcaagcnccataaaaaaaaaaaagagcncaaatgcattgggttttcaggtnta 300
tacattaagnngaacctttggcactaggaatcagggcgttttgtcacatagcnttaacac 360
atnttaaaaaattntgtantgtcaaagggatangaa 396
<210> 31
<211> 396
<212> DNA
<213> Homo sapien
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
11
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 31
gacgggccagggccatctggaaagggaactcggcttttccagaacgtggtggatcatctg 60
tcgggtgtgtggtgaacacgttcagttcatcagggcctacgctccgggaaggggccccca 120
gctgtggctctgccatgccgggctgtgtttgcagctgtccgagtctccatccgcctttag 180
aaaaccagccacttcttttcataagcactgacagggcccagcccacagccacaggtgcga 240
tcagtgcctcacgcaggcaaatgcactgaaacccaggggcacacncncgcagagtgaaca 300
gtgagttcccccgacagcccacgacagccaggactgccctccccaccccnccccgacccc 360
angancacggcacacanntcancctctnanctngct 396
<210> 32
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 32
cgactggcctcataccttgtctacacagtccctgcacagggttcctaacctgtggttagt 60
aaagaatgtcactttctaacaggtctggaagctccgagtttatcttgggaactcaagagg 120
agaggatcacccagttcacaggtatttgaggatacaaacccattgctgggctcggcttta 180
aaagtcttatctgaaattccttgtgaaacagagtttcatcaaagccaatccaaaaggcct 240
atgtaaaaataaccattcttgctgcactttatgcaaataatcaggccaaatataagacta 300
cagtttatttacaatttgtttttaccaaaaatgaggactanagagaaaaatggtgctcca 360
aagcttatcatacatttgtcattaagtcctagtctc 396
<210> 33
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
33
cctttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 60
tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 120
nngnnntntnnnnnannaaaaaaaaaaaaaaannnnnnnaaaaaaaannnnnnnnnnnnt 180
tttnnggggggnttttnanngnannttnnnnttnnnnnaaanccccnnngggnngggggg 240
nntnnnnnnggnaaaaaaannnnnnggggncnnnngggnccncncccnannnnnaaaann 300
nnnggnttttttnnttttnaaaaaaanngnnnnnnaacaaaantttttnnnnaanttttn 360
gggggaaannncccntttntttttttnnannnnnnn 396
<210> 34
<211> 396
<212> DNA
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
12
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 34
acggaccnagctggaggagctgggtgtggggtgcgttgggctggtggggaggcctagttn 60
gggtgcaagtangtctgattgagcttgtgttgtgctgaagggacagccctgggtctaggg 120
ganagagnccctgagtgtgagacccaccttccccngtcccagcccctcccanttccccca 180
gggacggccacttcctgntccccgacncaaccatggctgaagaacaaccgcaggtcgaat 240
tgttcntgaaggctggcagtgatggggccaagattgggaactgcccattctcccacagac 300
tgttnatggtactgtggctcaaggnagtcaccttcaatgttaccaccnntgacaccaaaa 360
ggcggaccnanacagtgcanaagctgtgcccanngg 396
<210> 35
<211> 396
<212> DNA
<213> Homo sapien
<400> 35
tcgaccaaaatcaaatctggcactcacaagccctggccgacccccaatgggttttaccac 60
tccccctctagaccctgtcttgcaaaatcctctccctagccagctagtattttctgggct 120
aaagactgtacaaccagttcctccattttatagaagtttactcactccaggggaaatggt 180
gagtcctccaacctccctttcaaccagtcccatcattccaaccagtggtaccatagagca 240
gcaccccccgccaccctctgagccagtagtgccagcagtgatgatggccacccatgagcc 300
cagtgctgacctggcacccaagaaaaagcccaggaagtcaagcatgcctgtgaagattga 360
gaaggaaattattgataccgccgatgagtttgatga 396
<210> 36
<211> 396
<212> DNA
<213> Homo sapien
<400> 36
tcgacgggaagagcctgctacggtggactgtgagactcagtgcactgtcctcctcccagc 60
gaccccacgctggaccccctgccggaccctccacccttcggcccccaagcttcccagggg 120
cttcctttggactggactgtccctgctcatccattctcctgccacccccagacctcctca 180
gctccaggttgccacctcctctcgccagagtgatgaggtcccggcttctgctctccgtgg 240
cccatctgcccacaattcgggagaccacggaggagatgctgcttgggggtcctggacagg 300
agcccccaccctctcctagcctggatgactacgtgaggtctatatctcgactggcacagc 360
ccacctctgtgctggacaaggccacggcccagggcc 396
<210> 37
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 37
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
13
cgacggtgtcagcaactggccatgccacagcacataaagattacagtgacaagaaaaaca 60
ttgtttgaggattcctttcaacagataatgagcttcagtccccaagatctgcgaagacgt 120
ttgtgggtgatttttccaggagaagaaggtttagattatggaggtgtagcaagagaatgg 180
ttctttcttttgtcacatgaagtgttgaacccaatgtattgcctgtttgaatatgcaggg 240
aaggataactactgcttgcagataaaccccgcttcttacatcaatccagatcacctgaaa 300
tattttcgttttattggcagatttattgccatggctctgttccatgggaaaattcataga 360
cacgggtttttctttnccattctataagcgtatctt 396
<210> 38
<211> 396
<212> DNA
<213> Homo sapien
<400> 38
cgaccaaaatgataaatagctttaagaatgtgctaatgataaatgattacatgtcaattt 60
aatgtacttaatgtttaataccttatttgaataattacctgaagaatatattttttagta 120
ctgcatttcattgattctaagttgcactttttacccccatactgttaacatatctgaaat 180
cagaatgtgtcttacaatcagtgatcgtttaacattgtgacaaagtttaatggacagttt 240
tttcccatatgtatatataaaataatgtgttttacaatcagtggcttagattcagtgaaa 300
tacagtaattcattcaattatgatagtatctttacagacattttaaaaataagttatttt 360
tatatgctaatattctatgttcaagtggaatttgga 396
<210> 39
<211> 396
<212> DNA
<213> Homo sapien
<400> 39
tcgaccaagaatagatgctgactgtactcctcccaggcgccccttccccctccaatccca 60
ccaaccctcagagccacccctaaagagatactttgatattttcaacgcagccctgctttg 120
ggctgccctggtgctgccacacttcaggctcttctcctttcacaaccttctgtggctcac 180
agaacccttggagccaatggagactgtctcaagagggcactggtggcccgacagcctggc 240
acagggcaagtgggacagggcatggccaggtggccactccagacccctggcttttcactg 300
ctggctgccttagaacctttcttacattagcagtttgctttgtatgcactttgttttttt 360
ctttgggtcttgttttttttttccacttagaaattg 396
<210> 40
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1)'. . (396)
<223> n = A,T,C or G
<400> 40
ttttttttttttttgttatttagtttttatttcataatcataaacttaactctgcaatcc 60
agctaggcatgggagggaacaaggaaaacatggaacccaaagggaactgcagcgagagca 120
caaagattctaggatactgcgagcaaatggggtggaggggtgctctcctgagctacagaa 180
ggaatgatctggtggttaanataaaacacaagtcaaacttattcgagttgtccacagtca 240
gcaatggtgatcttcttgctggtcttgccattcctggacccaaagcgctccatggcctcc 300
acaatattcatgccttctttcactttgccaaacaccacatgcttgccatccaaccactca 360
gtcttggcagtgcanatgaaaaactgggaaccattt 396
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
14
<210> 41
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
41
tcgacctcttgtgtagtcacttctgattctgacaatcaatcaatcaatggcctagagcac 60
tgactgttaacacaaacgtcactagcaaagtagcaacagctttaagtctaaatacaaagc 120
tgttctgtgtgagaattttttaaaaggctacttgtataataacccttgtcatttttaatg 180
tacaaaacgctattaagtggcttagaatttgaacatttgtggtctttatttactttgctt 240
cgtgtgtgggcaaagcaacatcttccctaaatatatattacccaaagnaaaagcaagaag 300
ccagattaggtttttgacaaaacaaacaggccaaaagggggctgacctggagcagagcat 360
ggtgagaggcaaggcatgagagggcaagtttgttgt 396
<210> 42
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 42
cttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 60
aaaanccnnannaanananggnaannnannaaaaaanncaaaccncntntanaaaangcc 120
nntntnagggggggggttcaaaaccaaanggnngntnggangnaaannnaaaanttnnnn 180
gggggnanaaanaaaaagggnngaaanntgacccnanaangaccngaaancccgggaaac 240
cnngggntanaaaaaaagntganccctaaanncccccgnaaaangggggaagggnaannc 300
caaatccnntgngggttgggggnggggaaaaaaaaaaccccnaaaaantgnaaaaaaccg 360
ggnttnaaanatttgggttcgggggntttntnttaa 396
<210> 43
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
43
ttttttttttttttgcttcactgctttatttttgaaatcacaagcaattcaaagtgatca 60
tcattgaggcttctgttaaaagttcttccaaagttgcccagttttaanattaaacaatat 120
tgcactttaagatgaactaacttttgggattctcttcaaagaaggaaagtattgctccat 180
ctgtgcttttcttanactaaaagcatactgcanaaaactctattttaaaaatcaacactg 240
cagggtacagtaacatagtaaagtacctgcctattttanaatcctanagaacatttcatt 300
gtaagaaactagcccattatttaagtgtccacagtatttttcatttcantggtccaagat 360
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
gccaaggttt ccaaacacaa tcttgttctc taatac 396
<210> 44
<211> 396
<212> DNA
<213> Homo sapien
<400> 44
gacctagttttacctcttaaatatctctgttcccttctaagttgtttgctgtgttttctt 60
cagagcaagaaggttatattttttaaaatttacttagtaatgcacattcaaaacacacat 120
caagtcttcaggataaagttcaaaaccgctgtcatggccccatgtgatctctccctcccc 180
tacccctctatcatttagtttcttctgcgcaagccactctggcttcctttcagttttgtg 240
gttcccgtttttagctagttcagtggttttcaatgggcatttcttgcctttttttttcta 300
aacgacaaatagaaatacatcttctttattatcctccaaatccaattcagaggtaatatg 360
ctccacctacacacaattttagaaataaattaaaaa 396
<210> 45
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 45
ttttttttttttttaaannttntaaatttttaatgaaannganttagaacaatgtattat 60
tnacatgtaaataaaaaaagagancataanccccatatnctcnnnaaaggaaggganacn 120
gcnggccntttatnagaanannnnncatataagaccccattaagaagaatctggatctaa 180
anacttncaaacaggagttcacagtangtgaacagcannccctaatcccactgatgtgat 240
gnttcanataaaatcancancgntgatcgggnatcnnancaatntgancggaanannact 300
gctcnatatntttnagganncngatgtggtcattttttacaaagataatggccacaccct 360
tccngnccgaatcgancnganctcccnnttctgtgn 396
<210> 46
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1)...(396)
<223> n = A,T,C or G
<400>
46
tttttttttttttttttttctganacagagtctcattctgttgcctaggctggattgcag 60
tggtgccatctcggctcactgcaacctccgcctcctgggttccanaaattctcctgcctc 120
agcctcccgggtagctgggactanaggcacacgccaccacgccaggctaatttttatatt 180
tttagtananatggcgtttcaccatgttgaccanactgatctcgaactcccgacctcgtg 240
atccacccacctcggcctcccaaagtgctgggattacaggcgtgaaaccaccaggcccgg 300
cctgaaatatctatttnttttcagattatttttaaaattccatttgatgaatcttttaaa 360
gtgagctananaaagtgngtgtgtacatgcacacac 396
<210> 47
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
16
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223 > n = A, T, C or G
<400> 47
tttttttttttttttttgctgttgccaactgtttattcagggccctgaacgggtggtgcg 60
tggacatgcaacacactcgggcccacagcagcgtgaccggccgctcccaagccccgggcg 120
cacaaccacagccaggagcagcccctgccaccactgggccaccgtccagggccccacagg 180
accagccgaaggtgccccgggccgaggccagctgggtcaggtgtacccctagcctggggt 240
tgag.tgaggagcggcacccccagtatcctgtgtaccccaagttgcccagnaggccgaggg 300
ggccttgggctccatctgcactggccaccccgtgccaagcatcacagctgcgtgagcagg 360
tttgtgtgtgagcgtgtggcggggcctggttgtccc 396
<210> 48
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A, T, C or G
<400> 48
ctgggcctgtgccgaagggtctgggcagatcttccaaagatgtacaaaatgtagaaattg 60
ccctcaagcaaatgcaaagatgctcaacacccttagtcatcaagaaaatgcaaatggaat 120
ccacagagagatactgcacactgacaaagatggtcgtattactaaaggtgaataaccagc 180
gcggggggcacgtggagtcactggaacatttgtgcaatgctggtgggaatgtcaacccgt 240
gcggccctctggaataagcctggcagctcctccaagagttacccgtgtgacccagcaatt 300
ccactcctagctccacccacaggaattgaaagcaaagacgcaaacagatgcctgtgcacc 360
aaagttcacggcagcatccttcgccatagtggnaan 396
<210> 49
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
49
accccaaaatgggaaaggaaaagactcatatnaacattgncgtnattggacacgtacatt 60
cggncaagtncaccactactggncatntgatntataaatgcggnggcatcgacanaanaa 120
ccatngnaanatttganaaggaggctgctgatatnggaaagggctccntcnantntgcct 180
gggtcttggatnaactgaaanctgancntgaacgtggnntcaccattgatatctncttgt 240
ggaaatntnagaccancanntactatgtnactatcattgatgccccaggacacaganact 300
ttatcnaaancatgattacnnggacatntanagctgactgtgctngcctgattgtngctg 360
ctggtgttggtgaatttgaanctggtatntccaana 396
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
17
<210> 50
<211> 396
<212> DNA
<213> Homo sapien
<400> 50
cgacttcttgctggtgggtggggcagtttggtttagtgttatactttggtctaagtattt 60
gagttaaactgcttttttgctaatgagtgggctggttgttagcaggtttgtttttcctgc 120
tgttgattgttactagtggcattaacttttagaatttgggctggtgagattaattttttt 180
taatatcccagctagagatatggcctttaactgacctaaagaggtgtgttgtgatttaat 240
tttttcccgttcctttttcttcagtaaacccaacaatagtctaaccttaaaaattgagtt 300
gatgtccttataggtcactacccctaaataaacctgaagcaggtgttttctcttggacat 360
actaaaaaatacctaaaaggaagcttagatgggctg 396
<210> 51
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
51
ttttttttttttcagcgnggatttattttatttcattttttactctcaaganaaagaana 60
gttactattgcaggaacagacatttttttaaaaagcgaaactcctgacacccttaaaaca 120
gaaaacattgttattcacataataatgnggggctctgtctctgccgacaggggctgggtt 180
cgggcattagctgtgccgtcgacaatagccccattcaccccattcataaatgctgctgct 240
acaggaagggaacagcggctctcccanagagggatccaccctggaacacgagtcacctcc 300
aaagagctgcgactgtttganaatctgccaanaggaaaaccactcaatgggacctggata 360
acccaggcccgggagtcatagcaggatgtggtactt 396
<210> 52
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 52
acctcgctaagtgttcgctacgcggggctaccggatcggtcggaaatggcagaggtggag 60
gagacactgaagcgactgcanagccagaagggagtgcagggaatcatcgtcgtgaacaca 120
gaaggcattcccatcaagagcaccatggacaaccccaccaccacccagtatgccagcctc 180
atgcacagnttcatcctgaaggcacggagcaccgtgcgtgacatcgacccccagaacgat 240
ctcaccttccttcgaattcgctccaagaaaaatgaaattatggttgcaccagataaagac 300
tatttcctgattgtgattcagaatccaaccgaataagccactctcttggctccctgtgtc 360
attccttaatttaatgccccccaagaatgttaatgt 396
<210> 53
<211> 396
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
18
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 53
tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 60
tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 120
tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 180
ttttttttttttttttttttttttttttttttttttttttttannttnttttttnttttn 240
cctttnttttaattcanaaaaagaanaagaaaanataanannnancnnannnnnnnnatn 300
ntncttnatantnnttnnnnnanngggnnngcgagnnnnnnnnnnnnnnnnntctnnnnt 360
tnnnnnncttgcnccccttnnnttngnnnnangcaa 396
<210> 54
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 54
ctcttggggctgctgggactcgcgtcggttggcgactcccggacgtaggtagtttgttgg 60
gccgggttctgaggccttgcttctctttacttttccactctaggccacgatgccgcagta 120
ccagacctgggaggagttcagccgcgctgccgagaagctttacctcgctgaccctatgaa 180
ggcacgtgtggttctcaaatataggcattctgatgggaacttgtgtgttaaagtaacaga 240
tgatttagtttgtttggtgtataaaacagaccaagctcaagatgtaaagaagattgagaa 300
attccacagtcaactaatgcgacttatggtagccaaggaagcccgcaatgttaccatgga 360
aactgantgaatggtttgaaatgaagactttgtcgt 396
<210> 55
<211> 396
<212> DNA
<213> Homo sapien
<400>
55
cgacggtttgccgccagaacacaggtgtcgtgaaaactacccctaaaagccaaaatggga 60
aaggaaaagactcatatcaacattgtcgtcattggacacgtagattcgggcaagtccacc 120
actactggccatctgatctataaatgcggtggcatcgacaaaagaaccattgaaaaattt 180
gagaaggaggctgctgagatgggaaagggctccttcaagtatgcctgggtcttggataaa 240
ctgaaagctgagcgtgaacgtggtatcaccattgatatctccttgtggaaatttgagacc 300
agcaagtactatgtgactatcattgatgccccaggacacagagactttatcaaaaacatg 360
attacagggacatctcaggctgactgtgctgtcctg 396
<210> 56
<211> 396
<212> DNA
<213> Homo sapien
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
19
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
56
ttttttttttttttttctcatttaacttttttaatgggtctcaaaattctgtgacaaatt 60
tttggtcaagttgtttccattaaaaagtactgattttaaaaactaataacttaaaactgc 120
cacacgcaaaaaanaaaaccaaagnggtccacaaaacattctcctttccttctgaaggtt 180
ttacgatgcattgttatcattaaccagtcttttactactaaacttaaatggccaattgaa 240
acaaacagttctganaccgttcttccaccactgattaanagtggggtggcaggtattagg 300
gataatattcatttagccttctgagctttctgggcanacttggngaccttgccagctcca 360
gcagccttnttgtccactgctttgatgacacccacc 396
<210> 57
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 57
cctttttttttttttttttttttttttttttttttttttttttttttttttnaaaanntt 60
ntttttgcaaanccnancaaaaanggnnggaangaaaaannggaaaaattntttttncnt 120
ntttgggaacnnnnagcccttnntttgaaaaaangnggncttaaaanngntgaannaaag 180
gnnanncccngntncttnnntttaaaaanaanggggnngnttttttttaaanaanatttt 240
ttttttccctaanancnncnanntgaaacnngncccnacnnctnncttnaaagggnnnaa 300
atnanangnnaaaaaanccctnancccccccccttanntttncnannananaaagncntt 360
ttgggncntgnaaaaanaancctttttnntgcnttn 396
<210> 58
<211> 396
<212> DNA
<213> Homo sapien
<400> 58
cgacctcaaatatgccttattttgcacaaaagactgccaaggacatgaccagcagctggc 60
tacagcctcgatttatatttctgtttgtggtgaactgattttttttaaaccaaagtttag 120
aaagaggtttttgaaatgcctatggtttctttgaatggtaaacttgagcatcttttcact 180
ttccagtagtcagcaaagagcagtttgaattttcttgtcgcttcctatcaaaatattcag 240
agactcgagcacagcacccagacttcatgcgcccgtggaatgctcaccacatgttggtcg 300
aagcggccgaccactgactttgtgacttaggcggctgtgttgcctatgtagagaacacgc 360
ttcacccccactccccgtacagtgcgcacaggcttt 396
<210> 59
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
<223> n = A,T,C or G
<400>
59
cttttttttttttttttttt~tcagnggaaaataacttttattganaccccaccaactgca 60
aaatctgttcctggcattaagctccttcttcctttgcaattcggtctttcttcagnggtc 120
ccatgaatgctttcttctcctccatggtctggaagcggccatggccaaacttggaggngg 180
tgtcaatgaacttaaggncaatcttctccanagcccgccgcttcntctgcaccancaagg 240
acttgcggagggngagcacccgcttnttggttcccaccacncagcctttcagcatgacaa 300
agtcattggtcacttcaccatagnggacaaagccacccaaagggttgatgctccttggca 360
aataggncatagtcacnggaggcattgtncttgatc 396
<210> 60
<211> 396
<212> DNA
<213> Homo sapien
<400>
60
acctcagctctcggcgcacggcccagcttccttcaaaatgtctactgttcacgaaatcct 60
gtgcaagctcagcttggagggtgatcactctacacccccaagtgcatatgggtctgtcaa 120
agcctatactaactttgatgctgagcgggatgctttgaacattgaaacagccatcaagac 180
caaaggtgtggatgaggtcaccattgtcaacattttgaccaaccgcagcaatgcacagag 240
acaggatattgccttcgcctaccagagaaggaccaaaaaggaacttgcatcagcactgaa 300
gtcagccttatctggccacctggagacggtgattttgggcctattgaagacacctgctca 360
gtatgacgcttctgagctaaaagcttccatgaaggg 396
<210> 61
<211> 396
<212> DNA
<213> Homo sapien
<400> 61
tagcttgtcggggacggtaaccgggacccggtgtctgctcctgtcgccttcgcctcctaa 60
tccctagccactatgcgtgagtgcatctccatccacgttggccaggctggtgtccagatt 120
ggcaatgcctgctgggagctctactgcctggaacacggcatccagcccgatggccagatg 180
ccaagtgacaagaccattgggggaggagatgactccttcaacaccttcttcagtgagacg 240
ggcgctggcaagcacgtgccccgggctgtgtttgtagacttggaacccacagtcattgat 300
gaagttcgcactggcacctaccgccagctcttccaccctgagcagctcatcacaggcaag 360
gaagatgctgccaataactatgcccgagggcactac 396
<210> 62
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 62
tcgacgtttcctaaagaaaaccactctttgatcatggctctctctgccagaattgtgtgc 60
actctgtaacatctttgtggtagtcctgttttcctaataactttgttactgtgctgtgaa 120
agattacagatttgaacatgtagtgtacgtgctgttgagttgtgaactggtgggccgtat 180
gtaacagctgaccaacgtgaagatactggtacttgatagcctcttaaggaaaatttgctt 240
ccaaattttaagctggaaagncactggantaactttaaaaaagaattacaatacatggct 300
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
21
ttttagaatt tcnttacgta tgttaagatt tgngtacaaa ttgaantgtc tgtnctganc 360
ctcaaccaat aaaatctcag tttatgaaan aaannn 396
<210> 63
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 63
t.tntttttttnttttntnttttntcnttgnttgnacngaacccggcgctnnttccccacn 60
nnnnacggccgcccntattcannnntncntcanntannnaccgcaccctcggactgcnnn 120
tngggccccgccgncnanncnccnncncccanttcnccgccgccgccgccgccttttttt 180
attggcnnccatnanaaccggggncacctcncangngcgccnaaantnggggcangactc 240
anagggggccatcaaccnccaagnncaanctgganctctacaaacggcctacgntttntg 300
nccatgngggtagggntttacccgcnatgatgannatgnnaanaactttnncaanccctt 360
tattaaccaatgnggtgnggagacggaacntggtta 396
<210> 64
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
64
tcgacgtcggggtttcctgcttcaacagtgcttggacggaacccggcgctcgttccccac 60
cccggccggccgcccatagccagccctccgtcacctcttcaccgcaccctcggactgccc 120
caaggcccccgccgccgctccagcgccgcgcagccaccgccgccgccgccgcctntnctt 180
agtcgccgccatgacgaccgcgtccacctcgcaggtgcgccagaactaccaccaggactc 240
agaggccgccatcaaccgccagatcaacctggagctctacgcctcctacgtttacctgtc 300
catgtcttactactttgaccgcgatgatgtggctttgaanaactttgccaaatactttct 360
tcccaatctcatgaggagaaggaacatgctganaaa 396
<210> 65
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 65
tttttttttt tttttttttt tttttnacca ataatgcttt tattttccac atcaanatta 60
atttatatgt tagttttagt acaagtacta aaatgtatac ttnttgccct aatagctaag 120
gnatacataa gcttcaccat acatnttgca nccncctgtc tgtcctatgt cattgttata 180
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
22
aatgtanana ttttaggaaa ctnttttatt caacctggga catntatact gtaggagtta 240
gcactgacct gatgtnttat ttaaaagtaa tgnatattac ctttacatat attccttata 300
tattnaaacg tatttccatg ttatccagct taaaatcaca tggnggttaa aagcatgagt 360
tctgagtcaa atctggactg aaatcctgat gctccc 396
<210> 66
<211> 396
<212> DNA
<213> Homo sapien
<400> 66
tcgacttttttttttccaggacattgtcataattttttattatgtatcaaattgtcttca 60
atataagttacaacttgattaaagttgatagacatttgtatctatttaaagacaaaaaaa 120
ttcttttatgtacaatatcttgtctagagtctagcaaatatagtacctttcattgcagga 180
tttctgcttaatataacaagcaaaaacaaacaactgaaaaaatataaaccaaagcaaacc 240
aaaccccccgctcaactacaaatgtcaatattgaatgaagcattaaaagacaaacataaa 300
gtaacttcagcttttatctagcaatgcagaatgaatactaaaattagtggcaaaaaaaca 360
aacaacaaacaacaaacaaaacaaaacaaacaaaca 396
<210> 67
<211> 396
<212> DNA
<213> Homo sapien
<400> 67
acgcttttgtccttcattttaactgttatgtcatactgttatgttgacatatttctttat 60
aagagaatagaggcaaaagtatagaactgaggatcatttgtatttttgagttggaaatta 120
tgaaacttcaccatattatgatcatacatattttgaagaacagactgaccaaagctcacc 180
tgttttttgtgttaggtgctttggctgaacttgattccagcccccttttccctttggtgt 240
tgtgtatgtctcttcatttcctctcaaatcttcaactcttgccccatgtctccttggcag 300
caggatgctggcatctgtgtagtcctcatactgtttactgataacccacaaattcatttt 360
catggcagacctaagctcagaccctgccttgtcctg 396
<210> 68
<211> 396
<212> DNA
<213> Homo sapien
<400>
68
acctgagtcctgtcctttctctctccccggacagcatgagcttcaccactcgctccacct 60
tctccaccaactaccggtccctgggctctgtccaggcgcccagctacggcgcccggccgg 120
tcagcagcgcggccagcgtctatgcaggcgctgggggctctggttcccggatctccgtgt 180
cccgctccaccagcttcaggggcggcatggggtccgggggcctggccaccgggatagccg 240
ggggtctggcaggaatgggaggcatccagaacgagaaggagaccatgcaaagcctgaacg 300
accgcctggcctcttacctggacagagtgaggagcctggagaccgagaaccggaggctgg 360
agagcaaaatccgggagcacttggagaagaagggac 396
<210> 69
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
23
<223> n = A,T,C or G
<400>
69
ntcncngnngntgtggtnntttttttaatttttatnttttctttttttttctngctagcn 60
cttnctttttttggaattncggtncctttttntntcnattttttngacaaaaanaacctn 1'20
ttntttnanaccanagnnnggnncacncntnnaatntnccccttttncgntngggagctn 180
cncnttnnncgccnacntcantcgagacngtncttttnnntnnancannntnngtncgtt 240
gncngcnttnntncannantnttccctatnnacntgnnntcncncatnnttggacnancn 300
cctagccttnccatnntttnnttntttntnnatnancctngaaaacntcngnntnttcnc 360
nncnttnccncncncnccttcntatgtncnatgncn 396
c210> 70
c211> 396
<212> DNA
<213> Homo sapien
c220>
c221> misc_feature
c222> (1). .(396)
c223> n = A,T,C or G
c400> 70
ttttttttttttttnttttttttttttttttttttttnttttttttttttttttttntnc 60
aannnntnaacttttaannggccnccngcnccccaanggggaccctgcttttgnnggcta 120
aatgccnnaaaactttggggnantnggtatnaaaccccnctttgcccnncannttncngg 180
ggggggggggtttttgnnggggaacangnanaacnttttnncnanggnatcaccaaaaan 240
aaagcccnnccctttttccnanngggggggggnggggggaaantcancccccanattgac 300
cttnatttcaaaanggggcttataatcctgggcntgganncttccctntacccgggggtt 360
gnccacnttttattanaggggnangnggatccccnt 396
c210> 71
<211> 396
<212> DNA
c213> Homo sapien
<220>
<221> misc_feature
c222> (1) . . (396)
c223> n = A,T,C or G
<400> 71
gcatctagagggccngtttantctagaggnccngnntaaacnnnnncatcnacctncnnt 60
gcncctgctngttgccncccntctgtgncttgcnnnncccnngagcgtnccttnaccnnn 120
gaangtgcctnnnnnactgannnnnncnnataanatgngganantncgtcgncattntnt 180
natnnggggtgatgctattctggggggtggggnggngnnatnnnatactnnggggacgtn 240
nnatnangagnnatntcnngnttntctnntgntttntggggggcnatnngnnntctntnn 300
ggactcntcgcncannnatcaatancttnattcngtgtanngtccgnccntagnncngcn 360
ngtactnnanngttgnnntcattactnttcgtnngg 396
<210> 72
<211> 396
<212> DNA
<213> Homo sapien
<220>
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
24
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
72
tntttttttttttctaaaacatnactntttattnnnnangntttntgaacctctnngcnt 60
natggtgagagtttgtctgattaataanaatnggannnttnannanangcntgnncgcaa 120
ngatggcnncnctgtatatcccaccatcccattacactntgaaccttttntttgattaat 180
aaaaggaaggnatgcggggaanggggaaagagaatgcttgaacattnccatgngnccttn 240
gacaaactttccaatggaggcnggaacnaannaccaccanncaactcccctttttgtaat 300
ttnnnaacttncaacnnctanctntttattttggcntccctggnngaaacagnctgtatn 360
annnnnaagnccntgagaacatccctggntnncnna 396
<210> 73
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 73
ntcaacntngactnctgtgaggnatggtgctgggngcntatgcngtgngnttttggatac 60
naccttatggacantngcnntcccnnggaangatnataatncttactgnagnnactnnaa 120
nnttccntntcnaaaangttnaaaancattggatgtgccacaatgatgacagtttatttg 180
ctactcttgagtgctataatgatgaagatcttanccaccattatcttaactgangcaccc 240
aanatggtganttggggaacatatanagtacacctaagttcacatgaagttgtttnttcc 300
caggnnctaaagagcaagcctaactcaagccattgncacacaggtgagacacctctattt 360
tgtacttctcacttttaagggattagaaaatagcca 396
<210> 74
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 74
cctttttttttttttttactgngaatatatactttttatttagtcatttttgtttacaat 60
tgaaactctgggaattcaaaattaacatccttgcccgtgagcttcttatagacaccanaa 120
aaagtttcaaccttgtgttccacattgttctgctgtgctttgtccaaatgaacctttatg 180
agccggctgccatctagtttgacgcggattctcttgcccacaatttcgcttgggaagacc 240
aagtcctcaaggatggcatcgtgcacagctgtcagagtacggctcctgggacgcttttgc 300
ttattttttgtacggctttttcgagttggcttaggcagaattctcctctgagcgataaag 360
acgacatgcttcccactgaactttttctccaattcg 396
<210> 75
<211> 396
<212> DNA
<213> Homo sapien
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 75
tttttttttttttntttttttttttttttttttttttnaantntaangggganggcccct 60
tttttttaaactngnccnttttnctttccttttttnaaaaggaaaaaaaaanntttnttt 120
ttcnttnaaaaaccctttttcccacnaacaaaaaaaaccnttccccntnccttttnnnna 180
aaaaaaaggggctnggnntttccccttanncaaaaaaccntntccnngggnaaaaaantt 240
ntcnccgggggggaaacnnntgggggtgtnnccnaaatttgggggccntcggaagggggg 300
nnccncncctaaagangtntttcaaaanaaaaacccccntcctnttntaaaaanaaaana 360
aaanaangnnngnnttttttntcnttnnccccccaa 396
<210> 76
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 76
acattcttcagaaatacagtgatgaaaattcattttgaaactcaaatattttcattttgg 60
atattctcctgtttttattaaaccagngattacncctggccntccctntaaatgttctag 120
gaaggcatgtctgttgtnntttnnnnaaaannaaattntttttttttngnnaaaccccaa 180
atcccantttatcaggaagttagncnaatgaaatggaaattggntaatggacaaaagcta 240
gcttgtaaaaaggaccacccnnccacnngnctttacccccttggttngttgggggaaaaa 300
ccatnnttaaccntntggnnaaaattgggnncntaaagtttncntggnnaacagtncntn 360
cngtattnaattgncnttatnggaaaatcngggatt 396
<210> 77
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
77
tttttttttttttttttttttttttttttttatcaacatttatatgctttattgaaagtt 60
ganaanggcaacagttaaatncngggacnccttacaattgtgtaaanaacatgcncanaa 120
acatatgcatataactactatacaggngatntgcaaaaacccctactgggaaatccattt 180
cattagttanaactgagcatttttcaaagtattcaaccagctcaattgaaanacttcagt 240
gaacaaggatttacttcagcgtattcagcagctanatttcaaattacncaaagngagtaa 300
ctgngccaaattcttaaaatttntttaggggnggtttttggcatgtaccagtttttatgt 360
aaatctatntataaaagtccacacctcctcanacag 396
<210> 78
<211> 396
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
26
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 78
agctggcnaaaggngnatgngctgcnangcgattangnnnggtaacgtcannggntnncc 60
agtgcangacnttgtaaaacgacggccacatgaattgtaatacgactcactatngggcgn 120
attgggccgtgnaggatngtgntcacactcgaatgtatnctggcngatncananngcttt 180
atngctnttgacggngnntnanccanctngggctttagggggtatcccctcgcccctgct 240
tcnttgatttgcacgggcnnctccganttccttcataataccngacgcttcnatccccta 300
gctcngacctntcantntnttcnntgggttntnnccgntcacngcttncccgnangntat 360
aatctnggctcctttngggatccattantctttact 396
<210> 79
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 79
caccaaccaaaacctggcgccgttggcatcgtagagtgaacacaacccaaaaacgatacg 60
ccatctgttctgccctggctgcctcagccctaccagcactggtcatgtctaaaggncatc 120
gtattgaggaagttcctgaacttcctttggtangttgaagataaagctgaaggctacaag 180
aagaccaangaagntgttttgctccttaanaaacttanacgcctggaatgatatcaaaaa 240
ngctatgcctctcagcgaatgagactgganangcaaaatgagaaaccntcnccgcatcca 300
gcgnaggggccgtgcatctctatnntgangatnntggnancnttcaaggccttcagaacc 360
tccctngaaatnctctnctttaangaaccaaactgn 396
<210> 80
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
80
tgtacataggcatcttattcactgcaccctgtcacacccagcaccccccgccccgcacat 60
tatttgaaagactgggaatttaatggttagggacagtaaatctacttctttttccaggga 120
cgactgtcccctctaaagttaaagtcaatacaagaaaactgtctatttttagcctaaagt 180
aaaggctgtgaagaaaattcattttacattgggtagacagtaaaaaacaagtaaaataac 240
ttgacatgagcacctttagatccttcccttcatggggctttgggcccagaatgacctttg 300
aggcctgtaaanggattgnaatttcctataagctgtatagtggagggattggngggtcat 360
ttgagtaagccctccaagatacnttcaatacctggg 396
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
27
<210> 81
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
81
gcagctgaagttcagcaggtgctgaatcgattctcctcggcccctctcattccacttcca 60
acccctcccattattccagtactacctcagcaatttgtgccccctacaaatgttagagac 120
tgtatacgccttcgaggtcttccctatgcagccacaattgaggacatcctgcatttcctg 180
ggggagttcgccacagatattcgtactcatggggttcacatggttttgaatcaccagggn 240
ccgccatcaggagatgcctttatccagatgaagtctgcggacagancatttatggctgca 300
cagaagtggcataaaaaaaacatgaaggacagatatgttgaagttttcagtgtcagctga 360
nganagaacattgnngtannngggggnactttaaat 396
<210> 82
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 82
gactcagaaatgtcagtctcatgaagttcaaaagatcgagaatgtttgctatcttggtgg 60
agcagccgcagccaagcaagtaacttgtaaaatgaggaatgccatcacccctcgagtgtc 120
catcccacataacttggggttagagcacaagcgttcccaggaactactcaccttaccatc 180
ttggccgtttcatttgcttccaccagttctggaaagaganggcctagaagttcaaaaaaa 240
aagtaggaaangtgcttttggagaaaatcacctgctcctcagaactgggcttacaanctg 300
ngaagtacnctatgtgccacctaatcctcatatatgacctcaagagacnccaataagcat 360
atttccaccacggaatgaccagtgctttgggtaana 396
<210> 83
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 83
tttgatttaaganatttattatttttttaaaaaaagcaacttccagggttgtcattgtac 60
aggttttgcccagtctcctatagcatggtatagtgataactgattttttataacaatgac 120
tcagaggcattgaagatccataactatcttctgaattatcacagaaagaagaaagttaga 180
agagtttaatgttaagtgtattaaaaatcatattctaattcttttaatttggttatctga 240
gtatgataatataggagagctcagataacaaggaaaaggcattggggtaagaacactcct 300
tcccacaggatggcattaacagactttttctgcatatgctttatatagttgccaactaat 360
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
28
tcacctttta cncagcttna ttttttttta ctnggg 396
<210> 84
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 84
tttttacagcaatttttttttattgatgtttaacctgtatacaaccatacccattttaag 60
ngtacagacaaatgaattttgacaaattcattcactcatctaatcatcactataaccatg 120
atacagatttttatcactccaaaagtccatcctgtgctcttttcaagtccatcctcctca 180
tctgataccccaagccaccattgttttgctttctggaactacagttttgggnttttagaa 240
tttcatatatggtngaatcataccatttgnnatttggggctgacgnctttcctccaataa 300
tggatttgagaattatctacattttgcatggatcctgggttatttataccaacnangggt 360
tattatgnaaaatnggaccacaatttggnggcanta 396
<210> 85
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1)...(396)
<223> n = A, T, C or G
<400>
85
cagtgaccgtgctcctacccagctctgctccacagcgcccacctgtctccgcccctcggc 60
ccctcgcccggctttgcctaaccgccacgatgatgttctcgggcttcaacgcagactacg 120
aggcgtcatcctcccgctgcagcagcgcgtccccggccggggatagcctctcttactacc 180
actcacccgcagactccttctccagcatgggctcgcctgcaacgcgcaggacttctgcac 240
ggacctggccgctccagtgccaacttcattccacggcactgcatctcgaccanccggact 300
tgcannggttggggaanccgcccttgtttctccgtggcccatctaanaccaaacccntca 360
ccttttcggagnccccncccctccgntgggnttact 396
<210> 86
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 86
ttttnnactg aatgtttaat acatttgnag gaacagaaga aatgcagtan ggattaanat 60
tttataatta gacattaatg taacagatgn ttcatttttc aaagaagntn cccccttntc 120
cctatctttt tttaatcttc cttanagcaa taantagtaa ttactatatt tgtggacaag 180
ctgctccact gtgntggaca gtaattatta aatctttatg tttcacatca ttattacctt 240
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
29
ccanaattct accttcattt ccctgcacag gttcactgga ctggntcaca ancaaattgn 300
actccactca antanaagag cccaaagaaa ttagagtaac gncnantcct atgaattana 360
gacccaaaga tttnaggngn tgattagaaa cataan 396
<210> 87
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 87
atggaggcgctggggaagctgaagcagttcgatgcctaccccaagactttggaggacttc 60
cgggtcaagacctgcgggggcgccaccgtgaccattgtcagtggccttctcatgctgcta 120
ctgttcctgtccgagctgcagtattacctcaccacggaggtgcatcctgagctctacgtg 180
gacaagtcgcggggagataaactgaagatcaacatcgatgtactttttccncacatgcct 240
tgtgcctatctgagtattgatgccatggatgtggccngagaacancagctggatgnggaa 300
cacaacctgtttaagccaccactagataaagatgcatcccngtgagctcanagctgagcg 360
gcatgagcttgngaaantcnaggtgaccgggtttga 396
<210> 88
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
88
tccagagcagagtcagccagcatgaccgagcgccgcgtccccttctcgctcctgcggggc 60
cccagctgggaccccttccgcgactggtacccgcatagccgctcttcgaccaggccttcg 120
ggctgccccggctgccggaggagtggtcgcagtggttaggcggcagcagctggccaggct 180
acgtgcgccccctgccccccgccgcatcgagagccccgcagtggccgcgcccgctacagc 240
cgcgcngctcagccggcaactcacancggggctcggagatccgggacactgcggaccgct 300
ngcgcgtgccctggatgtcaccactttngcccggacaactgacggtnanacaaggatggg 360
gggtgganannccngtaanccaagaangggnaggac 396
<210> 89
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 89
gagagaacag taaacatcca gccttagcat ctctcangag tactgcagat cttcattagc 60
tatattcaca tggagnaatg ctattcaacc tatttctctt atcaaaacta attttgtatt 120
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
ctttgaccaatgttcctaaattcactctgcttctctatctcaatctttttcccctttctc 180
atctttcctccttttttcagtttctaactttcactggttctttggaatgntttttctttc 240
atctcttttcttttacattttggggtgtcccctctcttttcttaccctctttctncatcc 300
ttcttnttcttttgaattggctgccctttatcntctcatctgctgncatcttcatttctc 360
ctccctcctntttccnntcattctactctctcccnt 396
<210> 90
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 90
gggcgccggcgcgcccccccacccccgccccacgtctcgtcgcgcgcgcgtccgctgggg 60
gcggggagcggtcgggccggcngcggtcggccggcggcagggtggtgcgntttcnttttn 120
nattnnccncnttcttcttnn~tnnncnnnctnntanncnntnncnttcncnnnntttnc 180
tntntcttnaccnnnttttntaatcntcttctncntnnnntctcttnnatntnttnctta 240
nttcctnnnntttnttctntcntttctcncctnnntctcnnnctcnncnctcnncatttt 300
nntnttttntnccttctnntcttnnttctnntnntnntttnnnnttctnttnntcatntt 360
ncctntnttactntcancttntatnnncctcntttt 396
<210> 91
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 91
ntntcctnnatttttnnntcnncttttttttnnaatttttctttnttttntttataaaaa 60
tcnncacntaaaacngcggaanaggggatttnttnttngggngtancncnnggccncaaa 120
naaccccaaaaatancccaaaatgcacaggnccngggnaaangaccnacntgggtntttt 180
ntttntnaacaaggggggttttaaagggnatnggnatcaaagggnataaantttaaacct 240
ttganaaattttttaanaggcttgccccccactttggnccccnccccncngnngggatcc 300
aattttttttcnttggggctcccngncccnnannttccgggttnntggncnntcctnntt 360
tttttttttttgccttcacccntnccattncntttt 396
<210> 92
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 92
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
31
ctntttnnntntttttttccccatcatccanaaatgggttttattctcagccgagggaca 60
gcaggactggtaaaaactgtcaggccacacggttgcctgcacagcacccccatgcttggt 120
agggggtgggagggatggcgggggctggntgnccacaggccgggcatgacaaggaggctc 180
actggaggtggcacactttggagtgggatgtcgggggacancttctttggtanttgggcc 240
acaagattcccaaggatancacnnnnactgattnccannctanagncaagcggntggcca 300
tntgtangnnnttntntatntgactatttatagatttttatanaacagggnaagggcata 360
ccncaaaagggnccaantttttaccnccgggcnccc 396
<210> 93
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 93
gctgccacagatctgttcctttgtccgtttttgggatccacaggccctatgtatttgaag 60
ggaaatgtgtatggctcagatcctttttgaaacatatcatacaggttgcagtcctgaccc 120
aagaacagttttaatggaccactatgagcccagttacataaagaaaaaggagtgctaccc 180
atgttctcatccttcagaagaatcctgcgaacggagcttcagtaatatatcgtggcttca 240
catgtgaggaagctacttaacactagttactctcacaatgaaggacctgnaatgaaaaat 300
ctgnttctaaccnagtcctntttanattttagngcanatccagaccancgncggtgctcg 360
agtaattctttcatgggacctttggaaaactttcag 396
<210> 94
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 94
tgccttaaccagtctctcaagtgatgagacagtgaagtaaaattgagtgcactaaacgaa 60
taagattctgaggaagtcttatcttctgcagtgagtatggcccaatgctttctgnggcta 120
aacagatgtaatgggaagaaataaaagcctacgtgttggtaaatccaacagcaagggaga 180
tttttgaatcataataactcatanngtgctatctgtcagtgatgccctcagagctcttgc 240
tgntagctggcagctgacgcttctangatagttagnttggaaatggtcttcataataact 300
acacaaggaaagtcanccnccgggcttatgaggaattggacttaataaatttagngngct 360
tccnacctaaaatatatcttttggaagtaaaattta 396
<210> 95
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
32
<400> 95
cctcccacccncttanttcatgagattcganaatgncacttntgtgctntttnctnnttn 60
tattctnacnatttctttcttggngcggnannaatcccntttttnngggcgnctctcccn 120
ncttntnntttcntggngctntcccttttcnnnnnaaacttntacnnngtttanaantnt 180
ttctgnangggggnntccnaaanantttttccncctncctnattccnctctnaannctcn 240
cnaattgtttcccccccccnntagnntattttttctaaaaaattaactccnacgganaaa 300
attttccctaaaatttcncctccanatttngaaaaaacncgcccgganctnntntncgaa 360
tntnaatttttnaaaaaaanttattttcatcnggnn 396
<210> 96
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 96
cctgggtaccaaatttctttatttgaaggaatggtacaaatcaaagaacttaagtggatg 60
ttttggacaacttatagaaaaggtaaaggaaaccccaacatgcatgcactgccttggcga 120
ccagggaagtcaccccacggctatggggaaattagcccgangcttaactttcattatcac 180
tgcttccaagggngtgcttg~gcaaaaaaatattccgccaaccaaatcgggcgctccatct 240
tgcccagttggtnccgggnccccaattcttggatgctttcncctcttnttccggaatgng 300
ctcatgaantcccccaannggggcattttgccagnggccntttngccattcnagnnggcc 360
tgatccattttttccaatgtaatgccncttcattgn 396
<210> 97
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1)...(396)
<223> n = A,T,C or G
<400> 97
ctcaccctcctcntnnttntcanaatattgngaacttnntnctgntcgaatcactggcat 60
taaaggancactagctaatggcactaaatttacnnactanggaaacttttttataatant 120
gcaaaaacatntnaaaaagantgnagttcgcccatttctgcttnggaaganctcttcact 180
tntaancccnnatgnngncctttgggtcaaaanctccgcgattattacngngttncccnc 240
tatttgnccttcctttntccccaangccncanatttcnnaactttnccntnaaatgcctt 300
tatttnatnncntttcnacnncttaannttccctttnaanaangatccctncttcaaatn 360
ntttcccngttcctngcattncccnnnnatttctct 396
<210> 98
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc feature
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
33
<222> (1)...(396)
<223> n = A,T,C or G
<400> 98
acagggacaatgaagcctttgaagtgccagtctatgaagaggccgtggtgggactagaat 60
cccagtgccgcccccaagagttggaccaaccaccccctacagcactgttgtgataccccc 120
agcacctgangaggaacaacctaccatccagaggggccaggaaaagccaaactggaacag 180
aggcgaatggctcagaggggtncatggccaagaaggaagccctggaagaacttcaatcac 240
cttcggtttcgggaccaccggcttgtgtccctgttctgactgcanaacttggcgcngtnc 300
cccattanaacctntgactcnncccttgctataagnctgttttggcccctgatgatgata 360
gggtttttatgangacacttgggcacccccttaatg 396
<210> 99
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 99
nttntttttccgncnaaagggcaagngtttncatctttcctgnccncncaananngggtn 60
tntgtgcntttnttttttcccaaaacccgggtnggggacaccttttgagganccactnnt 120
cntccggggcnnnnttttagaaggngnctaanaagcntcttgnngggggaaaaacatctt 180
tttgcncccnacatacccccaaggggggggggtgtctgggagganactaangacttttnt 240
tttttnnccncaaanaactganggcccccattgctccccccccantctttaaaaaacccc 300
ttcaatttccttgncnggnaaaaanggttggnaaaaaangagngngcntcnnttncnttt 360
natggaaggnaaaaggtttttggttgnaaaaccccg 396
<210> 100
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
100
ctaacacggtgaaaccctgtctctactaaaaatacaaaaaaattagccaggcgtggtggc 60
gggcacctgtagtcccagctgctcaggaagctgaggcaggagaatggcgtgaacccagaa 120
ggcggagcttgcagtgagctgagatcgtgtcagtgcactccagcctgggcgacagagcga 180
gactcccgctcaaaaaaaaaaaaaaaaagagaaaagaaaaagctgcagngagctgggaat 240
gggccctatcccctccttggggatcaatgagaccccttttcaaaanaaaaaaaaaaataa 300
tgngattttggnaacatatggcactggtgcttcnnggaattctgtttntnggcatgnccc 360
cctntgactgnggaaaaatccagcaggaggcccana 396
<210> 101
<211> 396
<212> DNA
<213> Homo sapien
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
34
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 101
agttataactcaacagttcatttatatgctgttcatttaacagttcatttaaacagttca 60
ttataactgtttaaaaatatatatgcttatagncaaaanntgttgtggcgnagttgttgc 120
cgcttatagctgagcattatttcttaaattcttgaatgttcttttggngggntnctaaaa 180
ccgtatatgatccattttnatgggaaacngaattcntnncattatcncaccttggaaata 240
cnnaacgtgggggaaaaaaatcattcccnccntccaaaactatacttcttttatctngan 300
nttcttgntcctgcncnggtttngaatatanctgggcaaanggntttnccaaatccntnt 360
acnntnctttgggaantancggcaantcntcncttt 396
<210> 102
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
102
actatacataagaacangctcacatgggaggctggaggtgggtacccagctgctgtggaa 60
cgggtatggacaggtcataaacctagagtcagngtcctgttggcctagcccatttcagca 120
ccctgccacttggagnggacccctctactcttcttagcgcctaccctcatacctatctcc 180
ctnctcccatctcctacggactggcgccaaatggctttcctgccaattttgggatcttct 240
ctggctctccagcctgcttactcctctatttttaaagggccaaacaaatcccttctcttt 300
ctcaaacacagtaatgnggcactgaccctaccacacctcatgaagggggcttgttgcttt 360
tatttgggcccgatctggggggggcaaaatattttg 396
<210> 103
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 103
ttgtgttgggactgctgataggaagatgtcttcaggaaatgctaaaattgggcaccctgc 60
cccaacttcaaagccacagctggtatgccanatggtcaggttaaagatatcaacctgctg 120
actacaaaggaaaatatggtggggtcttcttttaccctcttgacttccctttgngngccc 180
cccgagancattgctttccgngatagggcaaaanaaattaaaaaacttaactggccagtg 240
aatggggcttctgnggatctccttctggcattacatnggcaatccctaaaaaacaagang 300
actgggacccataacattcttttgnatcaaccgaagcccccattgttangatatngggct 360
taaangctgatnaagcatctcgtccgggcnttttat 396
<210> 104
<211> 396
<212> DNA
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 104
aagggagggcgcgccaagaccttcccactcgngcacactgggggcgccgacangacgcaa 60
cccagtccaacttggatacccttggntttagttctcggacacttcttttatctctccgtc 120
gcaacttgtcaagttctcaanactgtctctctgngntatcttttttcttcgctgctcttc 180
nncccccgacgtatttntcaaaangtctgcaattgttgnatacntnganctncaccactg 240
ttacnaggtcatnaatttcncntcaactctntnccncttgttccctgatatntcggccgg 300
ngncnccaattctgtattttnctcntcaacgntctcacttttncctcctccnggccactt 360
tctccccttccttattccggcnttgtttgccnccat 396
<210> 105
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 105
tcaatagccagccagtgttcatttttatccttgagcttttagtaaaaacttcctggnttt 60
atttttagtcattgggtcatacagcactaaagtctgctatttatggaaactaactttttt 120
gtttttaatccaggccaacatgtatgtaaattaaatttttagataattgattatctcttt 180
gtactacttgagatttgattatgagatgtgcatattgctttgggaagagctcgaggaagg 240
aaataattctctcctttggtttgaacctcaactagataaaccctaggaattgttaactgc 300
acaagnattttcattccacaaaacctgaggcagctcttttgccagagcgttcctgnaccc 360
ccccaccccacttgccttgggtctttanaangagcc 396
<210> 106
<211> 396
<212> DNA
<213> Homo sapien
<400>
106
gctgtgtagcacactgagtgacgcaatcaatgtttactcgaacagaatgcatttcttcac 60
tccgaagccaaatgacaaataaagtccaaaggcattttctcctgtgctgaccaaccaaat 120
aatatgtatagacacacacacatatgcacacacacacacacacacccacagagagagagc 180
tgcaagagcatggaattcatgtgtttaaagataatcctttccatgtgaagtttaaaatta 240
ctatatatttgctgatggctagattgagagaataaaagacagtaacctttctcttcaaag 300
ataaaatgaaaagcaattgctcttttcttcctaaaaaatgcaaaagatttacattgctgc 360
caaatcatttcaactgaaaagaacagtattgctttg 396
<210> 107
<211> 396
<212> DNA
<213> Homo sapien
<220>
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
36
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
107
ttcacagaacanggtggtttattatttcaatagcaaagagctgaaaaatgtcgggtccca 60
taaaggagcagaacctgacccagagcctgcagtacatttccaccccacaggggtgcaggc 120
tgggccaggcagggccaaaggcagcagaaatgggagtaagagactgtgcccactgagaag 180
ctctgctgggtgtgggcaggtgggcatganatgatgatgatgtagtgtaaggaccaggta 240
ggcaaaacctgtcaggnttgntgaatgtcanagtggatccaaaaggctgagggggtcgtc 300
anaaggccggnggncccncccttgcccgtatgggccttcaaaaagtatgcttgctcatcc 360
gttgtttnccccanggagctgccangganaaggctn 396
<210> 108
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 108
gcctgcttttgatgatgtctacagaaaatgctggctgagctgaacacatttgcccaattc 60
caggtgtgcacagaaaaccgagaatattcaaaattccaaatttttttcttaggagcaaga 120
agaaaatgtggccctaaagggggttagttgaggggtagggggtagtgaggatcttgattt 180
ggatctctttttatttaaatgtgaatttcaacttttgacaatcaaagaaaagacttttgt 240
tgaaatagctttactgcttctcacgtgttttggagaaaannatcanccctgcaatcactt 300
tttgnaactgncnttgattttcngcnnccaagctatatcnaatatcgtctgngtanaaaa 360
tgncctggncttttgaangaatacatgngtgntgct 396
<210> 109
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 109
ggccgtaggcagccatggcgcccagcccggaatggcatggtcttgaagccccacttccac 60
aaggactggcagcggcgcgtggccacgtggttcaaccagccggcccggaagatccgcaga 120
cgtaaggcccggcaagccaaggcgcgccgcatcgctccgcgccccgcgtcgggtcccatc 180
cggcccatcgtgcgctgcccacggttcggtaccacacgaagggcgcgccggcgcggnttc 240
agcctggaggagctcagggtggccggatttacaagaagnggccngacatcngtattcttg 300
ggatncnngaagnggaacaagtcacngagtccttgcagccacntcagcggntgatgacac 360
cgttcnaactcatctnttcccaagaaacctcngnnc 396
<210> 110
<211> 396
<212> DNA
<213> Homo sapien
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
37
<220>
<221> misc_feature
<222> (1). .(396)
<223 > n = A, T, C or G
<400>
110
nntgggctcctnncantnataataaaccngactcatacnccacaaggagatgaacaggan 60
tatgtncatnctgacgcggaaacagngcanggagctgaggaggngccaagatgagaccta 120
nnggccnnggtgggcgcattcccggnggagggggccactaaggantacgannntcnagcg 180
gctcttgnnggcngncctcctcacncctgnntattcgattgtcncnnatgncntcctatn 240
atnntcannattctntnntnatctcntntacnncntcncnttcatgnttacngntccctc 300
tcnttctnaccnttntctgnanctcctttctnnnnctttcatctntnttcngctttcttt 360
ctnnaatcntnntttaacntnntctnctttntnatt 396
<210> 111
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 111
taangancatnctggnttntgcctnnccgnctnattgantgttaaaggcaattntgtggn 60
tgtcccagngaatgncggctnattttctttccacattgngcncattcactcctcccactc 120
ttggcatgtngngacataagcanggtacataatngnaaaaatctgnatttctgatgccan 180
angggtanancntnttgnatntcattccattgatatacagccactnttttatttttgatc 240
ancggccttcggntcactgcncanggtacttgacctcagtgtcactattatgggntttgg 300
tttcnctcttttncnggccnttntntttcncacnttncancttncttnntnnaaaannna 360
nncactctctcttgctctctngatacnnngtctnaa 396
<210> 112
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 112
tcaacgtcaccaattactgccatttagcccacgagctgcgtctcagctgcatggagagga 60
aaaaggtccagattcgaagcatggatccctccgccttggcaagcgaccgatttaacctca 120
tactggcagataccaacagtgaccggctcttcacagtgaacgatgttaaagntggaggct 180
ccaagnatggtatcatcaacctgcaaagtctgaagacccctacgctcaaggtgttcatgc 240
acgaaaacctctacttcaccaaccggaaggtgaattcggggggctgggcctcgctgaatc 300
acttggattccacattctgctatgcctcatgggactcgcagaacttcaggctggccaccc 360
tgctcccaccatcactgntngncaatantcacccag 396
<210> 113
<211> 396
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
38
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 113
nnnnttnnnnnggagccttaatttcagagttttattgtattgcactaaaggaacagcagg 60
atggntatacaattttctctcattcagttttgaaaatctgtagtacctgcaaattcttaa 120
gaatacctttaccaccagattagaacagtaagcataataaccaatttcttaataagtaat 180
gtcttacaaataaaaacacatttaaaatagctttaaatgcattcttcacaagtaattcag 240
catatattttatatcatggttacttatgcttangaattnnagcaggatntttattctttt 300
gatggaaatatgggaaaactntattcatgcatatacanggataatattcagcgaagggaa 360
aatcccgtttttattttggnaatgattcatatataa 396
<210> 114
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 114
aaatgggacaacgtgattcttttgttttaaataaatactnagaacacggacttggctcct 60
acaagcatttggactctaaggnttagaactggagagtcttacccatgggccccncncagg 120
gacgccacggttccctcccaccccgngatcaagacacggaatcngntggcgatngttgga 180
tcgcnatgtgccccttatctatagccttcccnggncatntacangcaggatgcggntggg 240
anaactacaactgnaatntctcnaacggtnatggtccccaccgatnaagattctacctng 300
tcttttcntcccctggagtgtgagtgnnngaggaagaagcccttnccttacatcaccttt 360
tgnacttctgaacaagancaanacnatggccccccc 396
<210> 115
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
115
ccgcctggttcggcccgcctgcctccactcctgcctctaccatgtccatcagggtgaccc 60
agaagtcctacaaggtgtccacctctggcccccgggccttcagcagccgctcctacacga 120
gtgggcccggttcccgcatcagctcctcgagcttctcccgagtgggcagcagcaactttc 180
gcggtggcctggcggcggctatggtggggccagcggcatgggaggcatcacccgcagtta 240
cggcaaccagagcctgctgagccccttgcctggaggnggaccccaacatcaagccgngcg 300
cacccaggaaaaggagcagancaagaccctcaacaacaagnttgcttcttcatagacaag 360
ggaccggtccttgaacagcanaacaagatgntggag 396
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
39
<210> 116
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
116
atctcagtttactagctaagtgactttgggcaagggatttaacctctcgtccctcagttt 60
cctcctatgtaaaatgacaaggataatagtaccaacccaatgtagattaaatgagtttac 120
gaagtgttagaatagtgcttggcacattagtgctttacaactgctattttgattgttgtt 180
gtgggctctctcaaatgcattgtctctagatgccagtgacccaggtcaaaatttaccttt 240
aaccaagctgcatgtttcccagactgntgcacagtcctctaccctgaganaaagcttcca 300
cccaaggatacttttactttctgctggaaaactgatgagcaanggcaacangggacactt 360
atcgccaactggaaangagaaattcttccttttgct 396
<210> 117
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 117
aaacattttttaataaaattcctatagaaagctcagtcatagggcaaatactcagttctc 60
tttcccatatcaccgaggattgagagctcccaatattctttggagaataagcagtagttt 120
tgctggatgttgccaggactcagagagatcacccatttacacattcaaaccagtagttcc 180
tattgcacatattaacattacttgcccctagcaccctaaatatatggnacctcaacaaat 240
aacttaaagatttccgtggggcgcganaccatttcaatttgaactaatatccttgaaaaa 300
aatcacattattacaagntttaataaatacnggaagaagagctggcatttttctaanatc 360
tgaattcngacttggntttattccataaatacggtt 396
<210> 118
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
118
accnncacctgntnnnttttaacnattacaacttctttatatggcagtttttactgggng 60
cctaacactctctttactgnctcaagnggaagtccaaacaaatttcatttttgtagtaaa 120
aaatctttatttccaaaatgatttgttagccaaaagaactataaaccacctaacaagact 180
ttggaagaaagagacttgatgcttcttataaattccccattgcanacaaaaaataacaat 240
ccaacaagagcatggtacccattcttaccattaacctggntttaannctccaaancnnga 300
tttaaaaatgaccccactgggcccaatccaacatganacctaggggggnttgccttgatt 360
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
angaatcccc cttanggact ttatctnggc tganaa 396
<210> 119
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A, T, C or G
<400> 119
atggccagctcactttaaataccacctcaagactcatcgaaatgaccgctccttcatctg 60
tcctgcagaaggttgtgggaaaagcttctatgtgctgcagaggctgaaggtgcacatgag 120
gacccacaatggagagaagccctttatgtgccatgagtctggctgtggtaagcagtttac 180
tacagctggaaacctgaagaaccaccggcgcatccacacaggagagaaacctttcctttg 240
tgaagcccaangatgtggccgtcctttgctgagtattctancttcgaaaacatctggngg 300
ntactcangagagaaagcctcattantgccantctgngggaaaaccttctntcagagngg 360
angcaggaatgtgcatattaaaaagctnccttgnac 396
<210> 120
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 120
catgggtcagtcggtcctgagagttcgaagagggcacattcccaaagacattcccagtca 60
tgaaatgtagaagactggaaaattaagacattatgtaaaggtagatatggcttttagagt 120
tacattatgcttggcatgaataaggtgccaggaaaacagtttaaaattatacatcagcat 180
acagactgctgttagaaggtatgggatcatattaagataatctgcagctctactacgcat 240
ttattgttaattgagttacanangncattcannactgagtttataganccatattgctct 300
atctctgngnagaacatttgattccattgngaagaatgcagtttaaaatatctgaatgcc 360
atctagatgtattgtaccnaaaggggaaaaataaca 396
<210> 121
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 121
tttttttttt ttttttttaa aatcaagtta tgtttaataa acattaataa atgtttactt 60
aaaagggtta ataaacnttt actacatggc aaattatttt agctagaatg cttttggctt 120
caagncatan aaaccagatt cnaatgccct taaanaattt tnaaanatcc attgangggg 180
ataactgtaa tccccaaggg gaanagggtt gggtatgaca ggtacanggg gccagcccag 240
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
41
tnntnncana nncagactct taccntcttt ctgctgtgnc accctcaggc attggctcca 300
ttctcngggn tgcncatggg aagatggctt tggacntaac nacacccttt tgtncacgta 360
aaggccngat gcagggtcaa anagnttccn ccatnt 396
<210> 122
<211> 396
<212> DNA
<213> Homo sapien
<400>
122
gtcgacatggctgccctctgggctcccagaacccacaacatgaaagaaatggtgctaccc 60
agctcaagcctgggcctttgaatccggacacaaaaccctctagcttggaaatgaatatgc 120
tgcactttacaaccactgcactacctgactcaggaatcggctctggaaggtgaagctaga 180
ggaaccagacctcatcagcccaacatcaaagacaccatcggaacagcagcgcccgcagca 240
cccaccccgcaccggcgactccatcttcatggccaccccctgcggtggacggttgaccac 300
cagccaccacatcatcccagagctgagctcctccagcgggatgacgccgtccccaccacc 360
tccctcttcttctttttcatccttctgtctctttgt 396
<210> 123
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 123
gccctttttttttttttttttttcctagtgccaggtttattccctcacatgggtggttca 60
catacacagcacanaggcacgggcaccatggganagggcagcactcctgccttctgaggg 120
gatcttggcctcacggtgtaanaaggganaggatggtttctcttctgccctcactagggc 180
ctagggaacccagnagcaaatcccaccacgccttccatntctcagccaagganaagccac 240
cttggtgacgtttagttccaaccattatagtaagtgganaagggattggcctggtcccaa 300
ccattacagggtgaanatataaacagtaaaggaanatacagtttggatgaggccacagga 360.
aggagcanatgacaccatcaaaagcatatgcaggga 396
<210> 124
<211> 396
<212> DNA
<213> Homo sapien
<400> 124
gaccattgccccagacctggaagatataacattcagttcccaccatctgattaaaacaac 60
ttcctcccttacagagcatacaacagagggggcacccggggaggagagcacatactgtgt 120
tccaatttcacgcttttaattctcatttgttctcacaccaacagtgtgaagtgcgtggta 180
taatctccatttcaaaaccaaggaagcagcctcagagtggtcgagtgacacacctcacgc 240
aggctgagtccagagcttgtgctcctcttgattcctggtttgactcagttccaggcctga 300
tcttgcctgtctggctcagggtcaaagacagaatggtggagtgtagcctccacctgatat 360
tcaggctactcattcagtcccaaatatgtattttcc 396
<210> 125
<211> 396
<212> DNA
<213> Homo sapien
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
42
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
125
ccctttttttttttttttttttttttttttttttttactttgnaacaaaaatttattagg 60
attaagtcaaattaaaaaacttcatgcnccnccncttgtcatatttacctgaaatgacaa 120
agttatacttagcttgagngnaaaacttgngccccaaaaattntgtttggaaagcaaaaa 180
aataattgatgcncatagcagngggcctgatnccnccacagngaatgttgtttaaggnct 240
aacaaacaggggncancaaagcatacattacttttaagctttgggnccaaggaaaangtc 300
attccctacctccttcaaaagcaaactcatnatagcctgggcncctaggnctggagcctn 360
ttttttcgagtctaanatgaacatntggatttcaan 396
<210> 126
<211> 396
<212> DNA
<213> Homo sapien
<400> 126
cgcgtcgactcgcaagtggaatgtgacgtccctggagaccctgaaggctttgcttgaagt 60
caacaaagggcacgaaatgagtcctcaggtggccaccctgatcgaccgctttgtgaaggg 120
aaggggccagctagacaaagacaccctagacaccctgaccgccttctaccctgggtacct 180
gtgctccctcagccccgaggagctgagctccgtgccccccagcagcatctgggcggtcag 240
gccccacgacctggacacgctggggctacggctacagggcggcatccccaacggctacct 300
ggtcctagacctcagcatgcaagaggccctctcggggacgccctgcctcctaggacctgg 360
acctgttctcaccgtcctggcactgctcctagcctc 396
<210> 127
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 127
ttttttttttttggnggtaaaatgcaaatgttttaaaatatgtttattttgtatgtttta 60
caatgaatacttcagcaaagaaaataattataatttcaaaatgcaatccctggatttgat 120
aaatatcctttataatcgattacactaatcaatatctagaaatatacatagacaaagtta 180
gctaatgaataaaataagtaaaatgactacataaactcaatttcagggatgagggatcat 240
gcatgatcagttaagtcactctgccactttttaaaataatacgattcacatttgcttcaa 300
tcacataaacattcattgcaggagttacacggctaatcattgaaaattatgatctttgtt 360
agcttaaaagaaaattcagtttaatacaaagacatt 396
<210> 128
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc feature
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
43
<222> (1)...(396)
<223> n = A,T,C or G
<400> 128
gccctttttttttttttttaaaggcaaataaaataagtttattgggatgtaaccccatca 60
taaattgaggagcatccatacaggcaagctataaaatctggaaaatttaaatcaaattaa 120
attctgcttttaaaaaggtgccttaagttaaccaagcattttgataacacattcaaattt 180
aatatataaaaatagatgta.tcctggaagatataatgaanaacatgccatgtgtataaat 240
tcanaatacgctttttacacaaagaactacaaaaagttacaaagacagccttcaggaacc 300
acacttaggaaaagtgagccgagcagccttcacgcaaagcctccttcaaanaagtctcac 360
aaagactccagaaccagccgagtntgtgaaaaagga 396
<210> 129
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
129
gcccttttttttttttttttttttactcagacaggcaatatttgctcacatttattctct 60
tgcatcgtaaatagtagccaactcacaaaaataaagtatacaanaatgtaatatttttta 120
aaataagattaacagtgtaagaaggaaaatctcaaaaaaagcanatagacaatgtanaaa 180
attgaaatgaaatcccacagtaanaaaaaaaaaacanaaaagtgcctatttaanaattat 240
gctacatgtggaacttaactagaccattttaanaaagaccaatttctaatgcaaattttc 300
tgaggttttcanattttatttttaaaatatgttatagctacatgttgtcnacncggccgc 360
tcgagtctanagggcccgtttaaacccgctgatcag 396,
<210> 130
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 130
cgcccttttttttttttttttanngnacgtgnctttatttctggatgatataaaanaaaa 60
aacttaaaaaacaccccaaaccaaacaccaatggatccccaaagcgatgtgactccctct 120
tcccacccggataaatagagacttctgtatgtcagtctaccctcccgcccccataacccc 180
ctctgctatanacatactctgggtatatattactctactcggcaatagacatctcccgaa 240
aatagaattcctgccctgacacctgactcttccctggccgcatcanaccacccgccactg 300
tagcacactggtgtccttgccccctgtggtcagggccatgctgtcatcccacaanaaggc 360
cacatttgtcacatggctgctgtgtccaccgtactt 396
<210> 131
<211> 396
<212> DNA
<213> Homo sapien
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
44
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 131
gcccttttttttttttttttttttttttttttcagtttacacaaaaacnctttaattgac 60
agtatacnnttttccaaaatatnttttngtaanaaaatgcaataattattaactatagtt 120
tttacaaacaagtttntcantaaattccagtgtncttnaaaccccnnncnannaaaacat 180
atatgancccccagttcctgggcaaactgttgaacattcactgcanacaaaaagaccanc 240
nccaaanagtcatctgngncctccatgctgngtttgcaccaaacctgagggancagctag 300
ngaccgtgacaaaagctntgctacagttttactntngccctntntgcctcccccatnatg 360
tttccttggtccctcantcctgtnggagtaagttcc 396
<210> 132
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 132
cgcgtcgaccgcggccgtagcagccgggctggtcctgctgcgagccggcggcccggagtg 60
gggcggcgntatgtaccttccacattgagtattcagaaagaagtgatctgaactctgacc 120
attctttatggatacattaagtcaaatataagagtctgactacttgacacactggctcgg 180
tgagttctgctttttctttttaatataaatttattatgttggtaaatttagcttttggct 240
tttcactttgctctcatgatataagaaaatgtaggttttctctttcagtttgaattttcc 300
tattcagtaaaacaacatgctagaaaacaaacttttggaaaggcattgtaactatttttt 360
caaatagaaccataataacaagtcttgtcttaccct 396
<210> 133
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 133
ntattacccctcctggnnanntggnnatannctgcaaggngatnnncccgnngaacttca 60
ctgatnnnccaatnaaaactgctttaaanctgactgcacatatgaattntaatacttact 120
tngcgggaggggtggggcagggacagcaagggggaggattgggaanacaatagacaggca 180
tgctggggatgcngcgggctctatggcttctgangcgnaaagaaccagctggggctctag 240
ggggtatccccacgcgccctgtagcngcncattaaacgcggcgggtgtggnggttacttc 300
gcaaagngaccgatncacttgccagcgccctagctgcccgctcctttngctttcttccct 360
tcctttctcgccacnttnnccggctntccccgncaa 396
<210> 134
<211> 396
<212> DNA
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 134
ttttttttttttctgctttttatatgtttaaaaatctctcattctattgctgctttattt 60
aaagaaagattactttcttccctacaagatctttattaattgtaaagggaaaatgaataa 120
ctttacaatgganacacctggcanacaccatcttaaccaaagcttgaagttaacataacc 180
agtaatagaactgatcaatatcttgtgcctcctgatatggngtactaanaaaaacacaac 240
atcatgccatgatagtcttgccaaaagtgcataacctaaatctaatcataaggaaacatt 300
anacaaactcaaattgaaggacattctacaaagtgccctgtattaaggaattattcanag 360
taaaggagacttaaaagacatggcaacaatgcagta 396
<210> 135
<211> 396
<212> DNA
<213> Homo sapien
<400> 135
gcgtcgacgctggcagagccacaccccaagtgcctgtgcccagagggcttcagtcagctg 60
ctcactcctccagggcacttttaggaaagggtttttagctagtgtttttcctcgctttta 120
atgacctcagccccgcctgcagtggctagaagccagcaggtgcccatgtgctactgacaa 180
gtgcctcagcttccccccggcccgggtcaggccgtgggagccgctattatctgcgttctc 240
tgccaaagactcgtgggggccatcacacctgccctgtgcagcggagccggaccaggctct 300
tgtgtcctcactcaggtttgcttcccctgtgcccactgctgtatgatctgggggccacca 360
ccctgtgccggtggcctctgggctgcctcccgtggt 396
<210> 136
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
136
ttatgcttccggctcgtntgttgtgtggaattgtgagcggataacaatttcacacaggaa 60
acagctatgaccatgattacgccaagctatttaggtgacactatagaatactcaagctat 120
gcatcaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattc 180
gcggncgntcnantctagagggcccgtttaaacccgctgatcagcctcgactgtgccttc 240
tagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgc 300
cactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtg 360
tcattctattctggggggtggggtggggcaggacan 396
<210> 137
<211> 396
<212> DNA
<213> Homo sapien
<220>
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
46
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 137
ttttttttttttctgctttgtacttgagtttatttcacaaaaccacggagaaagatactg 60
aaatggagctctttccagcctccaagcaaggaggccccagcagccagtctccagcccctt 120
gagccctttttgttaggcccacacccaaaagagganaaccagtgtgtgcgcgaaggtaca 180
tggcaaggcacttttgaaaacatcccagtttaccgnggtgaaattgaacttactctgaaa 240
cagatgaaaagggacatgcaaaattgctgagcacatggaggtgtttgttagtaggtgaaa 300
atcatgtcctgggtataacccagcttctccaggttagggtgagccgccgtctggatcagt 360
ggtggcgggccacacaccaggatgagcgtggacttc 396
<210> 138
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
138
ccctttttttttttttttacaaatgagaaaaatgtttattaagaaaacaatttagcagct 60
ctcctttanaattttacagactaaagcacaacccgaaggcaattacagtttcaatcatta 120
acacactacttaaggngcttgcttactctacaactggaaagttgctgaagtttgtgacat 180
gccactgtaaatgtaagtattattaaaaattacaaattgtttggtgattattttgatgac 240
ctcttgagcagcagctccccccaanaatgcancaatggtatgtggctcaccagctccata 300
tcggcaaaattcgtggacataatcatctttcaccattacagataaaccatattcctgaag 360
gaagccagtgagacaagacttcaactttcctatatc 396
<210> 139
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 139
ccgcccttttttttttttttttcacaaaagcactttttatttgaggcaaanagaagtctt 60
gctgaaaggattccagttccaagcagtcaaaactcaaccgttagnggcactattttgacc 120
tggtanattttgcttctctttggtcanaaaagggtattcaggttgtactttccccagcag 180
ggtaaaaagaagggcaaagcaaactggaananacttctactctactgacagggctnttga 240
natccaacatcaagctanacacnccctcgctggccactctacaggttgctgtcccactgc 300
tgagtgacacaggcc.atactacatttgcaaggaaaaaaatgaggcaanaaacacaggtat 360
aggtcacttggggacgagcaggcaaccacagcttca 396
<210> 140
<211> 396
<212> DNA
<213> Homo sapien
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
47
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
140
tttttttttttttttttttttttttttctcatttaacttttttaatgggnctcaaaattn 60
tgngacaaatttttggtcaagttgtttccattaaaaagtnctgattttaaaaactaataa 120
cttaaaactgccncncccaaaaaaaaaaaccaaaggggtccacaaaacattntcctttcc 180
ttntgaaggntttacnatgcattgttatcattaaccagtnttttactactaaacttaaan 240
ggccaattgaaacaaacagttntganaccgttnttccnccactgattaaaagnggggggg 300
caggtattagggataatattcatttanccttntgagctttntgggcanacttggngacct 360
tgccagctccagcagccttnttgtccactgntttga 396
<210> 141
<211> 396
<212> DNA
<213> Homo sapien
<400>
141
acgccgagccacatcgctcagacaccatggggaaggtgaaggtcggagtcaacggatttg 60
gtcgtattgggcgcctggtcaccagggctgcttttaactctggtaaagtggatattgttg 120
ccatcaatgaccccttcattgacctcaactacatggtttacatgttccaatatgattcca 180
cccatggcaaattccatggcaccgtcaaggctgagaacgggaagcttgtcatcaatggaa 240
atcccatcaccatcttccaggagcgagatccctccaaaatcaagtggggcgatgctggcg 300
ctgagtacgtcgtggagtccactggcgtcttcaccaccatggagaaggctggggctcatt 360
tgcaggggggagccaaaagggtcatcatctctgccc 396
<210> 142
<211> 396
<212> DNA
<213> Homo sapien
<400> 142
acgcaggagaggaagcccagcctgttctaccagagaacttgcccaggtcagaggtctgcg 60
tagaagcccttttctgagcatcctctcctctcctcacacctgccactgtcctctgcgttg 120
ctgtcgaattaaatcttgcatcaccatggtgcacttctgtggcctactcaccctccaccg 180
ggagccagtgccgctgaagagtatctctgtgagcgtgaacatttacgagtttgtggctgg 240
tgtgtctgcaactttgaactacgagaatgaggagaaagttcctttggaggccttctttgt 300
gttccccatggatgaagactctgctgtttacagctttgaggccttggtggatgggaagaa 360
aattgtagcagaattacaagacaagatgaaggcccg 396
<210> 143
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 143
tttttttttt tttccatana aaataggatt tattttcaca tttaaggnga acacaaatcc 60
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
48
atgttccanaaatgttttatgcataacacatcatgagtagattgaatttctttaacacac 120
anaaaaatcaaagcctaccaggaaatgcttccctccggagcacaggagcttacaggccac 180
ttntgttagcaacacaggaattcacattgtctaggcacagctcaagngaggtttgttccc 240
aggttcaactgctcctacccccatgggccctcctcaaaaacgacagcagcaaaccaacag 300
gcttcacagtaaccaggaggaaagatctcagngggggaaccttcacaaaagccctgagtt 360
gtgtttcaaaagccaagctctggggtctgnggcctg 396
<210> 144
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 144
tttttttttttttcgctctttggtctgacaagaaaagagttttaggtgtgtgaagtaggg 60
tgggaaaaaaggtcagtttcaaattcagtaacatatggtaacactaagttaggctgctgc 120
attcttttctttgggtacttaagccagctggcacttccactttgtaaccaattatattat 180
gatcaacaactaatcagttagttcctcagcttcaactgaanagttcctgattacctgatg 240
aaggacatacttgctctggcttcaattagcatgctgtcaagcatccctctccatgcttaa 300
catggcaacacaaaacccaagagtccttctntttttttcattagccatgaataaacactc 360
acaaaggggaagagtagacactgcttttagtaaacg 396
<210> 145
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 145
tttttttttttttttttcaatggatccgttagctttactactaanatcttgctganatca 60
nanaagggcttctgggcaggctgagcactgggggtgtgcaacatggtaactctgaataan 120
anaaaccctgagttttactgggcaaanaaanaacaagnggtaggtatgatttctgaacct 180
ggaaatagcgaaaatgaaggaaattccaaaagcgcgtatttccaaataatgacaggccag 240
caagaggacaccaaacctntanaaagaggtattntttcttccagctactgatggctttgg 300
catcccacaggcacattcctttggccttcaggatcttanatgcanatgtgganagtcaag 360
aggtaggctgactctgagtcttcagctaaattcttt 396
<210> 146
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
49
<400>
146
ttttttttttttttcattagcaaggaaggatttattttttcttttgaggggagggcggaa 60
cagccgggatttttggaacactacctttgtctttcactttgttgtttgtgtgttaacacn 120
aataaatcanaagcgactttaaatctcccttcgcaggactgtcttcacgtatcagngcan 180
acaanaaaacagtggctttacaaaaaanatgttcaagtaggctgcactttgcctctgngg 240
gtgaggcacactgngggananacaaggtcccctgnaaccagaggngggaaggacanagct 300
ggctgactccctgctctcccgcattctctcctccatgtgttttgaanagggaagcaacat 360
gttgaggtctgatcatttctacccagggaacctgtt 396
<210> 147
<211> 396
<212> DNA
<213> Homo sapien
<400> 147
acggggaagccaagtgaccgtagtctcatcagacatgagggaatgggtggctccagagaa 60
agcagacatcattgtcagtgagcttctgggctcatttgctgacaatgaattgtcgcctga 120
gtgcctggatggagcccagcacttcctaaaagatgatggtgtgagcatccccggggagta 180
cacttcctttctggctcccatctcttcctccaagctgtacaatgaggtccgagcctgtag 240
ggagaaggaccgtgaccctgaggcccagtttgagatgccttatgtggtacggctgcacaa 300
cttccaccagctctctgcaccccagccctgtttcaccttcagccatcccaacagagatcc 360
tatgattgacaacaaccgctattgcaccttggaatt 396
<210> 148
<211> 396
<212> DNA
<213> Homo sapien
<400> 148
acgtcccatgattgttccagaccatgactcttcctggttgtgggtttgttacagagcagg 60
agaagcagaggttatgacagttatgcagactttccccctcctttttctcttttctcttcc 120
ccttgcttttccactgtttcttcctgctgccacctgggccttgaattcctgggctgtgaa 180
gacatgtagcagctgcagggtttaccacacgtgggagggcagcccagtactgtccctctg 240
ccttccccactttgagaatatggcagcccctttcattcctggcttggggtaggggagacc 300
attgaagtagaagcctcaaagcagacttttccctttactgtgtgtactccaggacgaaga 360
aggaagatcatgcttgatacttagattggttttccc 396
<210> 149
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
149
tttttttttttttaaagagtcacattttattcaatgcctatttgtacatgttactagcaa 60
taaactcttttatctttaattttgagaagttttacaaatacagcaaagcagaatgactaa 120
tagagccggtaaccaggacacagatttggaaaaataggtctaattggttgttacactgtg 180
tttatgtcatacatttcgcttatttttatcaaanaaaaatcagaatttataaaatgttaa 240
ttaaaaggaaaacattctgagtaaatttagtcccgtgtttcttcctccaaatctntttgt 300
tctacactaacaggtcaggataagtatggatggggaggctggaaaaagggcatccttccc 360
catgcggtccccagagccaccctctccaagcaggac 396
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
<210> 150
<211> 396
<212> DNA
<213> Homo sapien
<400> 150
acgcctctcttcagttggcacccaaacatctggattggcaaatcagtggcaagaagttcc 60
agcatctggacttttcagaattgatcttaagtctactgtcatttccagatgcattatttt 120
acaactgtatccttggaaatatatttctagggagaatattattgaagaaaatgttaatag 180
cctgagtcaaatttcagcagacttaccagcatttgtatcagtggtagcaaatgaagccaa 240
actgtatcttgaaaaacctgttgttcctttaaatatgatgttgccacaagctgcattgga 300
gactcattgcagtaatatttccaatgtgccacctacaagagagatacttcaagtctttct 360
tactgatgtacacatgaaggaagtaattcagcagtt 396
<210> 151
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
151
acaaaatgcccagcctacagagtctgagaaggaaatttataatcaggtgaatgtagtatt 60
aaaagatgcagaaggcatcttggaggacttgcagtcatacagaggagctggccacgaaat 120
acgagaggcaatccagcatccagcanatgagaagttgcaagagaaggcatggggtgcagt 180
tgttccactagtaggcaaattaaagaaattttacgaattttctcagaggttagaagcagc 240
attaagaggtcttctgggagccttaacaagtaccccatattctcccacccagcatctana 300
gcgagagcaggctcttgctaaacagtttgcanaaattcttcatttcacactccggtttga 360
tgaactcaagatgacaaatcctgccatacagaatga 396
<210> 152
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 152
acgcagcgctcggcttcctggtaattcttcacctcttttctcagctccctgcagcatggg 60
tgctgggccctccttgctgctcgccgccctcctgctgcttctctccggcgacggcgccgt 120
gcgctgcgacacacctgccaactgcacctatcttgacctgctgggcacctgggtcttcca 180
ggtgggctccagcggttcccagcgcgatgtcaactgctcggttatgggaccacaagaaaa 240
aaaagtagnggtgtaccttcagaagctggatacagcatatgatgaccttggcaattctgg 300
ccatttcaccatcatttacaaccaaggctttgagattgtgttgaatgactacaagtggtt 360
tgccttttttaagtataaagaagagggcagcaaggt 396
<210> 153
<211> 396
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
51
<212> DNA
<213> Homo sapien
<400> 153
ccagagacaacttcgcggtgtggtgaactctctgaggaaaaacacgtgcgtggcaacaag 60
tgactgagacctagaaatccaagcgttggaggtcctgaggccagcctaagtcgcttcaaa 120
atggaacgaaggcgtttgcggggttccattcagagccgatacatcagcatgagtgtgtgg 180
acaagcccacggagacttgtggagctggcagggcagagcctgctgaaggatgaggccctg 240
gccattgccgccctggagttgctgcccagggagctcttcccgccactcttcatggcagcc 300
tttgacgggagacacagccagaccctgaaggcaatggtgcaggcctggcccttcacctgc 360
ctccctctgggagtgctgatgaagggacaacatctt 396
<210> 154
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 154
acagcaaacctcctcacagcccactggtcctcaagaggggcnacntcttcacacatcanc 60
acaactacgcattgcctccctncactcggaaggactatcctgctgccaagagggtcaagt 120
tggacagtgtcagagtcctgagacagatcagcaacaaccgaaaatgcaccagccccaggt 180
cctcggacaccgaggagaatgtcaagaggcgaacacacaacgtcttggagcgccagagga 240
ggaacgagctaaaacggagcttttttgccctgcgtgaccagatcccggagttggaaaaca 300
atgaaaaggcccccaaggtagttatccttaaaaaagccacagcatacatcctgtccgtcc 360
aagcagaggagcaaaagctcatttctgaagaggact 396
<210> 155
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
155
tttttttttttgaananacaggtctttaatgtacggagtctcacaaggcacaaacaccct 60
caccaggaccaaataaataactccacggttgcaggaaggcgcggtctggggaggatgcgg 120
catctgagctctcccagggctggtgggcgagccgggggtctgcagtctgtgaggggcctc 180
ctgggtgtgtccgggcctctanagcgggtccagtctccaggatggggatcgctcactcac 240
tctccgagtcggagtagtccgccacgagggaggagccganactgcaggggtgccgcgtgt 300
cgggggtgtcagctgcctcctgggaggagcctgctggcnacaggggcttgtcctgacggc 360
tcccttcctgccccctcgggctgctgcacttggggg 396
<210> 156
<211> 396
<212> DNA
<213> Homo sapien
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
52
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400> 156
gaaggggggcngggcaggggcggaatgtananattantgccatgattgaagatttaagaa 60
acgtgagattcaggattttcaccacatccccatttagttagcttgctcgtttggctggtg 120
caaatgccagatggattatgaacaatgacagtaaattaatgcaacataatcaggtaatga 180
tgccaagcgtatctggtgttccaggtattgtacctttaccggaacaaatcagtaaatcca 240
caatccctggcacctgttaggcagctattaacctagtaaatgctcccccatcccatctca 300
atcagcaangacaatcaaaaacatttgctttnagtggcaggaacactggtacatttttac 360
ttgctccaagggctgtgccaacgctccctctctctg 396
<210> 157
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
157
tttttttttttttttggggaatgtaaatcttttattaaaacagttgtctttccacagtag 60
taaagctttggcacatacagtataaaaaataatcacccaccataattataccaaattcct 120
nttatcaactgcatactaagtgttttcaatacaattttttccgtataaaaatactgggaa 180
aaattgataaataacaggtaananaaagatatttctaggcaattactaggatcatttgga 240
aaaagtgagtactgnggatatttaaaatatcacagtaacaagatcatgcttgttcctaca 300
gtattgcgggccanacacttaagtgaaagcanaagtgtttgggtgactttcctacttaaa 360
attttggncatatcatttcaaaacatttgcatcttg 396
<210> 158
<211> 396
<212> DNA
<213> Homo sapien
<400> 158
tttccgaagacgggcagcttcagagaagaggattattcgggagattgctggtgtggccca 60
tagactctttggcatagactctttcgcaggcagccactctgagtgtggccagttctataa 120
ccatccccaaactagctggagcctgatggataggaacgggtagtctgtcctcttccccat 180
aaaaatgttccaaaaagttatctccagagagagtcccttatgaagacagttgccaagctg 240
tattctcattctttaaaccaatacccaggtcagggctagttcacactagcactgttaggg 300
acatggtgtggctagaaatgaattgagtgtgacttctccctacaaccccaggcccaggga 360
taggaggaggcagaggggtgcctggagtttctgcac 396
<210> 159
<211> 396
<212> DNA
<213> Homo sapien
<400> 159
tccgcgcgtt gggaggtgta gcgcggctct gaacgcgctg agggccgttg agtgtcgcag 60
gcggcgaggg cgcgagtgag gagcagaccc aggcatcgcg cgccgagaag gccgggcgtc 120
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
53
cccacactgaaggtccggaaaggcgacttccgggggctttggcacctggcggaccctccc 180
ggagcgtcggcacctgaacgcgaggcgctccattgcgcgtgcgcgttgaggggcttcccg 240
cacctgatcgcgagaccccaacggctggtggcgtcgcctgcgcgtctcggctgagctggc 300
catggcgcagctgtgcgggctgaggcggagccgggcgtttctcgccctgctgggatcgct 360
gctcctctctggggtcctggcggccgaccgagaacg 396
<210> 160
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 160
ggaaaccttctcaactaagagaacatcatttctggcaaactatttttgttagctcacaat 60
atatgtcgtacactctacaatgtaaatagcactganccacancttacagaaggtaaaaag 120
angnataanaacttcctttacaaaananttcctgttgttcttaatactccccattgctta 180
tganaattntctatangtctctcangantgttcgcacccatttcttttntaacttctact 240
aaaaanccatttacattgnanagtgtacnacntatatttgngagctaacaaaaaatngtt 300
ttccnganatgatgttcttttagtttnaganggttcnnncaanttnctactccngcccgc 360
cactgnncnccacatttnnnnaattacaccncacng 396
<210> 161
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400>
161
tttttgtttgattatttttattataatgaaattaaacttatgactattacagtatgctca 60
gcttaaaacatttatgagtactgcaaggactaacagaaacaggaaaaatcctactaaaaa 120
tatttgttgatgggaaatcattgtgaaagcaaacctccaaatattcatttgtaagccata 180
agaggataagcacaaccatatgggaggagataaccagtctctcccttcatatatattctt 240
ttttatttcttggtataccttcccaaaacananacattcaacagtagttagaatggccat 300
ctcccaacattttaaaaaaactgcnccccccaatgggtgaacaaagtaaagagtagtaac 360
ctanagttcagctgagtaagccactgtggagcctta 396
<210> 162
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 162
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
54
ttttttttttttttttttttttttttttttttnggggnccaaatttttttntttgaagga 60
angggacaaannaaaaaacttaaggggntgttttggnncnacttanaaaaaagggaaagg 120
aaaccccaacatgcatgccctnccttggggaccanggaanncnccccncnggtntgggga 180
aantaacccnaggnttaactttnattatcactgncncccagggggggcttnnaaaaaaaa 240
nnttcccccaanccaaantngggnncncccattttncncaanttggncnccnggncnccc 300
nattttttgangggtttcnccngcncattnagggaangggnntcaannaaaccncncaaa 360
ngggggnnatttttntcangggccnatttgngcnnt 396
<210> 163
<211> 396
<212> DNA
<213> Homo sapien
<400>
163
cactgtccggctctaacacagctattaagtgctacctgcctctcaggcactctcctcgcc 60
cagtttctgaggtcagacgagtgtctgcgatgtcttcccgcactctattcccccagcctc 120
tttctgctttcatgctcagcacatcatcttcctaggcagtctcttccccaaagtctcacc 180
ttttcttccaatagaaaattccgcttgacctttggtgcactgcccacttcccagctccac 240
tggcccaagtctgagccggaggcccttgttttgggggcggggggagagttggatgtgatt 300
gcccttgaagaacaaggctgacctgagaggttcctggcgccctgaggtggctcagcacct 360
gcccagggtaggcctggcatgaggggttaggtcagc 396
<210> 164
<211> 396
<212> DNA
<213> Homo sapien
<400> 164
gacacgcggcggtgtcctgtgttggccatggccgactacctgattagtgggggcacgtcc 60
tacgtgccagacgacggactcacagcacagcagctcttcaactgcggagacggcctcacc 120
tacaatgactttctcattctccctgggtacatcgacttcactgcagaccaggtggacctg 180
acttctgctctgaccaagaaaatcactcttaagaccccactggtttcctctcccatggac 240
acagtcacagaggctgggatggccatagcaatggcgcttacaggcggtattggcttcatc 300
caccacaactgtacacctgaattccaggccaatgaagttcggaaagtgaagaaatatgaa 360
cagggattcatcacagaccctgtggtcctcagcccc 396
<210> 165
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
<223> n = A,T,C or G
<400>
165
ttttttttttttttttttttttttttcangggncactgaggctttttattttgancncaa 60
aaccnccggggatctancctgnggccnccccggaaatnacncnaggctcacatnactnta 120
aacncttgggggaaagggaggcaaaaaaaacaatgacttgggccaattncncnactgcaa 180
agntananctgccaacagggctccagggagcttggnttntgtaaaanttntaaggaagcg 240
gnncnaactccncggggggggggcnctaactancagggacccctgcaagngttggncggg 300
ggcctcaacctgcctgagctnacncaaggggnggggtntntntanccaacaggggaccna 360
agggcttgcctncccacagnttacttggccaagggg 396
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
<210> 166
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 166
ttttttcaaattcagagcatttttattaaaagaacaaaatattaaggcacaaaatacatc 60
aatttttcaaatgaaaacccttcaaacggttatgtcctacattcaacgaaacttcttcca 120
aattacggaataatttaactttttaaaatanaaaaatacaagttcttaaatgcctaaaat 180
ttctccccaaataaatgttttcttagttttaatgaagtctcttcatgcagtactgagctc 240
caatattataatgtncacttccttaaaaatctagttttgccacttatatacattcaatat 300
gtttaaccagtatattaaccagtatattaaccaatatgttaaacttcttttaagtataag 360
gcttggtattttgtattgcttattgcatgctttgat 396
<210> 167
<211> 396
<212> DNA
<213> Homo sapien
<400> 167
tggcggcagcggcggtggcggtggctgagcagaggacccggcgggcggcctcgcgggtca 60
ggacacaatgtttgcacgaggactgaagaggaaatgtgttggccacgaggaagacgtgga 120
gggagccctggccggcttgaagacagtgtcctcatacagcctgcagcggcagtcgctcct 180
ggacatgtctctggtgaagttgcagctttgccacatgcttgtggagcccaatctgtgccg 240
ctcagtcctcattgccaacacggtccggcagatccaagaggagatgacgcaggatgggac 300
gtggcgcacagtggcaccccaggctgcagagcgggcgccgctcgaccgcttggtctccac 360
ggagatcctgtgccgtgcagcgtgggggcaagaggg 396
<210> 168
<211> 396
<212> DNA
<213> Homo sapien
<400>
168
taggatggtaagagtattataaggattggtacaaggcatgatgagtccttttgcttttag 60
gcttttgacttctggttttagactttctttagcttctgttgttagacaacattgtgcaag 120
cttggtttttataagtttgcatggattaaactgaacttaatgaaattgtccctcccccca 180
aattctcagcacaatttttaggcccacaaggagtcaagcacctcaaggagatcttcagtt 240
tgaacttggtgtagacacagggatactgatgaatcaatattcaaattagctgttacctac 300
ttaagaaagagaggagaccttggggatttcgaggaagggttcataagggagattttagct 360
gagaaataccatttgcacagtcaatcacttctgacc 396
<210> 169
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1). .(396)
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
56
<223> n = A,T,C or G
<400> 169
tttttttttttttcanaattaaattctttaatacaaaatgcttttttttttttaaaanat60
atctgtatttctttgncgttgttnaaaaataaatatgtnctacggaatatntcnaaaaac120
tgcnctaaaaacaaanacgngatgttaatatcttttccccncaattnttacggataaaca180
gtanccccnataaataaatgatancnaatnttaaaattaaaaaagganananatttagta240
tgnaaaattctctattttttcttggtttggttttncntataaaaaacanaatagcaatgt300
ntnttttatcanaatcccntntntncctaaacntttttttttttntttncccccnaatnc360
aagnngccaaanatntntntagnatgnanatgtntn 396
<210> 170
<211> 396
<212> DNA
<213> Homo sapien
<400> 170
tgagaagtac catgccgcttctgcagaggaacaggcaaccatcgaacgcaacccctacac 60
catcttccat caagcactgaaaaactgtgagcctatgattgggctggtacccatcctcaa 120
gggaggccgt ttctaccaggtccctgtacccctacccgaccggcgtcgccgcttcctagc 180
catgaagtgg atgatcactgagtgccgggataaaaagcaccagcggacactgatgccgga 240
gaagctgtca cacaagctgctggaggctttccataaccagggccccgtgatcaagaggaa 300
gcatgacttg cacaagatggcagaggccaaccgtgccctggcccactaccgctggtggta 360
gagtctccag gaggagcccagggccctctgcgcaag 396
<210> 171
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 171
ggtcctcgtcgtggtgagcgcagccactcaggctggtcctgggggtggggctgtagggga60
aagtgctaaagccgctgagtgaagtaagaactctgctagagaggaaaatgggcttgcttt120
catcatcatcctnctcagctggtggggtcaagtgggaagttctgtcactgggatctggtt180
cagtgtctcaagaccttgccccaccacggaaagcctttttcacntaccccaaaggacttg240
gagagatgttagaagatggntctnaaanattcctctgcnaatntgtttttagctatcaag300
tggcttccccccttaancaggnaaaacatgatcagcangttgctcggatggaaaaactan360
cttggtttgnnaaaaaanctggaggcttgacaatgg 396
<210> 172
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 172
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
57
agccttgggccaccctcttggagcatctggctgtcgaattcttgtgaccctgttacacac 60
actggagagaatgggcagaagtcgtggtgttgcagccctgtgcattgggggtgggatggg 120
aatagcaatgtgtgttcagagagaatgaattgcttaaactttgaacaacctcaatttctt 180
tttaaactaataaagtactaggttgcaatatgtgaaaaaaaaaaaaaaagggcggccgnt 240
cnantntanagggcccnttnaaacccgttgatcaacctcgactgtgccttctagttgcca 300
gccatctgttgttngcccctcccccgtgnctttcttgaccttgaaaggggccccncccct 360
gtctttcctaanaaaaangaagaantnnccttccnt 396
<210> 173
<211> 396
<212> DNA
<213> Homo sapien
<220>
<221> misc_feature
<222> (1) . . (396)
<223> n = A,T,C or G
<400> 173
aagcatgtggatatgtttagctacgtttactcacagccagcgaactgacattaaaataac 60
taacaaacagattcttttatgtgatgctggaactcttgacagctataattattattcaga 120
aatgactttttgaaagtaaaagcagcataaagaatttgtcacaggaaggctgtctcagat 180
aaattatggtaaaattttgcaggggacannctttttaagacttgcacaattnccggatcc 240
tgcnctgactttggaaaaggcatatatgtnctagnggcatgganaatgccccatactcat 300
gcatgcaaattaaacaaccaagtttgaatctttttgggggngngctatnctttaacccng 360
tacnggcnttattatntaangnccctgnnncntgtg 396
<210> 174
<211> 924
<212> DNA
<213> Homo sapiens
<400> 174
cctgacgacc cggcgacggc gacgtctctt ttgactaaaa gacagtgtcc agtgctccag 60
cctaggagtc tacggggacc gcctcccgcg ccgccaccat gcccaacttc tctggcaact 120
ggaaaatcat ccgatcggaa aacttcgagg aattgctcaa agtgctgggg gtgaatgtga 180
tgctgaggaa gattgctgtg gctgcagcgt ccaagccagc agtggagatc aaacaggagg 240
gagacacttt ctacatcaaa acctccacca ccgtgcgcac cacagagatt aacttcaagg 300
ttggggagga gtttgaggag cagactgtgg atgggaggcc ctgtaagagc ctggtgaaat 360
gggagagtga gaataaaatg gtctgtgagc agaagctcct gaagggagag ggccccaaga 420
cctcgtggac cagagaactg accaacgatg gggaactgat cctgaccatg acggcggatg 480
acgttgtgtg caccagggtc tacgtccgag agtgagtggc cacaggtaga accgcggccg 540
aagcccacca ctggccatgc tcaccgccct gcttcactgc cccctccgtc ccaccccctc 600
cttctaggat agcgctcccc ttaccccagt cacttctggg ggtcactggg atgcctcttg 660
cagggtcttg ctttctttga cctcttctct cctcccctac accaacaaag aggaatggct 720
gcaagagccc agatcaccca ttccgggttc actccccgcc tccccaagtc agcagtccta 780
gccccaaacc agcccagagc agggtctctc taaaggggac ttgagggcct gagcaggaaa 840
gactggccct ctagcttcta ccctttgtcc ctgtagccta tacagtttag aatatttatt 900
tgttaatttt attaaaatgc.ttta 924
<210> 175
<211> 3321
<212> DNA
<213> Homo sapiens
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
58
<400> 175
atgaagattt tgatacttgg tatttttctg tttttatgta gtaccccagc ctgggcgaaa 60
gaaaagcatt attacattgg aattattgaa acgacttggg attatgcctc tgaccatggg 120
gaaaagaaac ttatttctgt tgacacggaa cattccaata tctatcttca aaatggccca 180
gatagaattg ggagactata taagaaggcc ctttatcttc agtacacaga tgaaaccttt 240
aggacaacta tagaaaaacc ggtctggctt gggtttttag gccctattat caaagctgaa 300
actggagata aagtttatgt acacttaaaa aaccttgcct ctaggcccta cacctttcat 360
tcacatggaa taacttacta taaggaacat gagggggcca tctaccctga taacaccaca 420
gattttcaaa gagcagatga caaagtatat ccaggagagc agtatacata catgttgctt 480
gccactgaag aacaaagtcc tggggaagga gatggcaatt gtgtgactag gatttaccat 540
tcccacattg atgctccaaa agatattgcc tcaggactca tcggaccttt aataatctgt 600
aaaaaagatt ctctagataa agaaaaagaa aaacatattg accgagaatt tgtggtgatg 660
ttttctgtgg tggatgaaaa tttcagctgg tacctagaag acaacattaa aacctactgc 720
tcagaaccag agaaagttga caaagacaac gaagacttcc aggagagtaa cagaatgtat 780
tctgtgaatg gatacacttt tggaagtctc ccaggactct ccatgtgtgc tgaagacaga 840
gtaaaatggt acctttttgg tatgggtaat gaagttgatg tgcacgcagc tttctttcac 900
gggcaagcac tgactaacaa gaactaccgt attgacacaa tcaacctctt tcctgctacc 960
ctgtttgatg cttatatggt ggcccagaac cctggagaat ggatgctcag ctgtcagaat 1020
ctaaaccatc tgaaagccgg tttgcaagcc tttttccagg tccaggagtg taacaagtct 1080
tcatcaaagg ataatatccg tgggaagcat gttagacact actacattgc cgctgaggaa 1140
atcatctgga actatgctcc ctctggtata gacatcttca ctaaagaaaa cttaacagca 1200
cctggaagtg actcagcggt gttttttgaa caaggtacca caagaattgg aggctcttat 1260
aaaaagctgg tttatcgtga gtacacagat gcctccttca caaatcgaaa ggagagaggc 1320
cctgaagaag agcatcttgg catcctgggt cctgtcattt gggcagaggt gggagacacc 1380
atcagagtaa ccttccataa caaaggagca tatcccctca gtattgagcc gattggggtg 1440
agattcaata agaacaacga gggcacatac tattccccaa attacaaccc ccagagcaga 1500
agtgtgcctc cttcagcctc ccatgtggca cccacagaaa cattcaccta tgaatggact 1560
gtccccaaag aagtaggacc cactaatgca gatcctgtgt gtctagctaa gatgtattat 1620
tctgctgtgg atcccactaa agatatattc actgggctta ttgggccaat gaaaatatgc 1680
aagaaaggaa gtttacatgc aaatgggaga cagaaagatg tagacaagga attctatttg 1740
tttcctacag tatttgatga gaatgagagt ttactcctgg aagataatat tagaatgttt 1800
acaactgcac ctgatcaggt ggataaggaa gatgaagact ttcaggaatc taataaaatg 1860
cactccatga atggattcat gtatgggaat cagccgggtc tcactatgtg caaaggagat 1920
tcggtcgtgt ggtacttatt cagcgccgga aatgaggccg atgtacatgg aatatacttt 1980
tcaggaaaca catatctgtg gagaggagaa cggagagaca cagcaaacct cttccctcaa 2040
acaagtctta cgctccacat gtggcctgac acagagggga cttttaatgt tgaatgcctt 2100
acaactgatc attacacagg cggcatgaag caaaaatata ctgtgaacca atgcaggcgg 2160
cagtctgagg attccacctt ctacctggga gagaggacat actatatcgc agcagtggag 2220
gtggaatggg attattcccc acaaagggag tgggaaaagg agctgcatca tttacaagag 2280
cagaatgttt caaatgcatt tttagataag ggagagtttt acataggctc aaagtacaag 2340
aaagttgtgt atcggcagta tactgatagc acattccgtg ttccagtgga gagaaaagct 2400
gaagaagaac atctgggaat tctaggtcca caacttcatg cagatgttgg agacaaagtc 2460
aaaattatct ttaaaaacat ggccacaagg ccctactcaa tacatgccca tggggtacaa 2520
acagagagtt ctacagttac tccaacatta ccaggtgaaa ctctcactta cgtatggaaa 2580
atcccagaaa gatctggagc tggaacagag gattctgctt gtattccatg ggcttattat 2640
tcaactgtgg atcaagttaa ggacctctac agtggattaa ttggccccct gattgtttgt 2700
cgaagacctt acttgaaagt attcaatccc agaaggaagc tggaatttgc ccttctgttt 2760
ctagtttttg atgagaatga atcttggtac ttagatgaca acatcaaaac atactctgat 2820
caccccgaga aagtaaacaa agatgatgag gaattcatag aaagcaataa aatgcatgct 2880
attaatggaa gaatgtttgg aaacctacaa ggcctcacaa tgcacgtggg agatgaagtc 2940
aactggtatc tgatgggaat gggcaatgaa atagacttac acactgtaca ttttcacggc 3000
catagcttcc aatacaagca caggggagtt tatagttctg atgtctttga cattttccct 3060
ggaacatacc aaaccctaga aatgtttcca agaacacctg gaatttggtt actccactgc 3120
catgtgaccg accacattca tgctggaatg gaaaccactt acaccgttct acaaaatgaa 3180
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
59
gacaccaaat ctggctgaat gaaataaatt ggtgataagt ggaaaaaaga gaaaaaccaa 3240
tgattcataa caatgtatgt gaaagtgtaa aatagaatgt tactttggaa tgactataaa 3300
cattaaaaga gactggagca t 3321
<210> 176
<211> 487
<212> DNA
<213> Homo sapiens
<400> 176
gaaatacttt ctgtcttatt aaaattaata aattattggt ctttacaaga cttggataca 60
ttacagcaga catggaaata taattttaaa aaatttctct ccaacctcct tcaaattcag 120
tcaccactgt tatattacct tctccaggaa ccctccagtg gggaaggctg cgatattaga 180
tttccttgta tgcaaagttt ttgttgaaag ctgtgctcag aggaggtgag aggagaggaa 240
ggagaaaact gcatcataac tttacagaat tgaatctaga gtcttccccg aaaagcccag 300
aaacttctct gcagtatctg gcttgtccat ctggtctaag gtggctgctt cttccccagc 360
catgagtcag tttgtgccca tgaataatac acgacctgtt atttccatga ctgctttact 420
gtatttttaa ggtcaatata ctgtacattt gataataaaa taatattctc ccaaaaaaaa 480
aaaaaaa 487
<210> 177
<211> 3999
<212> DNA
<213> Homo sapiens
<400> 177
caagattcca catttgatgg ggtgactgac aaacccatct tagactgctg tgcctgcgga 60
actgccaagt acagactcac attttatggg aattggtccg agaagacaca cccaaaggat 120
taccctcgtc gggccaacca ctggtctgcg atcatcggag gatcccactc caagaattat 180
gtactgtggg aatatggagg atatgccagc gaaggcgtca aacaagttgc agaattgggc 240
tcacccgtga aaatggagga agaaattcga caacagagtg atgaggtcct caccgtcatc 300
aaagccaaag cccaatggcc agcctggcag cctctcaacg tgagagcagc accttcagct 360
gaattttccg tggacagaac gcgccattta atgtccttcc tgaccatgat gggccctagt 420
cccgactgga acgtaggctt atctgcagaa gatctgtgca ccaaggaatg tggctgggtc 480
cagaaggtgg tgcaagacct gattccctgg gacgctggca ccgacagcgg ggtgacctat 540
gagtcaccca acaaacccac cattccccag gagaaaatcc ggcccctgac cagcctggac 600
catcctcaga gtcctttcta tgacccagag ggtgggtcca tcactcaagt agccagagtt 660
gtcatcgaga gaatcgcacg gaagggtgaa caatgcaata ttgtacctga caatgtcgat 720
gatattgtag ctgacctggc tccagaagag aaagatgaag atgacacccc tgaaacctgc 780
atctactcca actggtcccc atggtccgcc tgcagctcct ccacctgtga caaaggcaag 840
aggatgcgac agcgcatgct gaaagcacag ctggacctca gcgtcccctg ccctgacacc 900
caggacttcc agccctgcat gggccctggc tgcagtgacg aagacggctc cacctgcacc 960
atgtccgagt ggatcacctg gtcgccctgc agcatctcct gcggcatggg catgaggtcc 1020
cgggagaggt atgtgaagca gttcccggag gacggctccg tgtgcacgct gcccactgag 1080
gaaacggaga agtgcacggt caacgaggag tgctctccca gcagctgcct gatgaccgag 1140
tggggcgagt gggacgagtg cagcgccacc tgcggcatgg gcatgaagaa gcggcaccgc 1200
atgatcaaga tgaaccccgc agatggctcc atgtgcaaag ccgagacatc acaggcagag 1260
aagtgcatga tgccagagtg ccacaccatc ccatgcttgc tgtccccatg gtccgagtgg 1320
agtgactgca gcgtgacctg cgggaagggc atgcgaaccc gacagcggat gctcaagtct 1380
ctggcagaac ttggagactg caatgaggat ctggagcagg tggagaagtg catgctccct 1440
gaatgcccca ttgactgtga gctcaccgag tggtcccagt ggtcggaatg taacaagtca 1500
tgtgggaaag gccacgtgat tcgaacccgg atgatccaaa tggagcctca gtttggaggt 1560
gcaccctgcc cagagactgt gcagcgaaaa aagtgccgca tccgaaaatg ccttcgaaat 1620
ccatccatcc aaaagctacg ctggagggag gcccgagaga gccggcggag tgagcagctg 1680
aaggaagagt ctgaagggga gcagttccca ggttgtagga tgcgcccatg gacggcctgg 1740
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
tcagaatgca ccaaactgtg cggaggtgga attcaggaac gttacatgac tgtaaagaag 1800
agattcaaaa gctcccagtt taccagctgc aaagacaaga aggagatcag agcatgcaat 1860
gttcatcctt gttagcaagg gtacgagttc cccagggctg cactctagat tccagagtca 1920
ccaatggctg gattatttgc ttgtttaaga caatttaaat tgtgtacgct agttttcatt 1980
tttgcagtgt ggttcgccca gtagtcttgt ggatgccaga gacatccttt ctgaatactt 2040
cttgatgggt acaggctgag tggggcgccc tcacctccag ccagcctctt cctgcagagg 2100
agtagtgtca gccaccttgt actaagctga aacatgtccc tctggagctt ccacctggcc 2160
agggaggacg gagactttga cctactccac atggagaggc aaccatgtct ggaagtgact 2220
atgcctgagt cccagggtgc ggcaggtagg aaacattcac agatgaagac agcagattcc 2280
ccacattctc atctttggcc tgttcaatga aaccattgtt tgcccatctc ttcttagtgg 2340
aactttaggt ctcttttcaa gtctcctcag tcatcaatag ttcctgggga aaaacagagc 2400
tggtagactt gaagaggagc attgatgttg ggtggctttt gttctttcac tgagaaattc 2460
ggaatacatt tgtctcaccc ctgatattgg ttcctgatgc ccccccaaca aaaataaata 2520
aataaattat ggctgcttta tttaaatata aggtagctag tttttacacc tgagataaat 2580
aataagctta gagtgtattt ttcccttgct tttgggggtt cagaggagta tgtacaattc 2640
ttctgggaag ccagccttct gaactttttg gtactaaatc cttattggaa ccaagacaaa 2700
ggaagcaaaa ttggtctctt tagagaccaa tttgcctaaa ttttaaaatc ttcctacaca 2760
catctagacg ttcaagtttg caaatcagtt tttagcaaga aaacattttt gctatacaaa 2820
cattttgcta agtctgccca aagccccccc aatgcattcc ttcaacaaaa tacaatctct 2880
gtactttaaa gttattttag tcatgaaatt ttatatgcag agagaaaaag ttaccgagac 2940
agaaaacaaa tctaagggaa aggaatatta tgggattaag ctgagcaagc aattctggtg 3000
gaaagtcaaa cctgtcagtg ctccacacca gggctgtggt cctcccagac atgcatagga 3060
atggccacag gtttacactg ccttcccagc aattataagc acaccagatt cagggagact 3120
gaccaccaag ggatagtgta aaaggacatt ttctcagttg ggtccatcag cagtttttct 3180
tcctgcattt attgttgaaa actattgttt catttcttct tttataggcc ttattactgc 3240
ttaatccaaa tgtgtaccat tggtgagaca catacaatgc tctgaataca ctacgaattt 3300
gtattaaaca catcagaata tttccaaata caacatagta tagtcctgaa tatgtacttt 3360
taacacaaga gagactattc aataaaaact cactgggtct ttcatgtctt taagctaagt 3420
aagtgttcag aaggttcttt tttatattgt cctccacctc catcattttc aataaaagat 3480
agggcttttg ctcccttgtt cttggaggga ccattattac atctctgaac tacctttgta 3540
tccaacatgt tttaaatcct taaatgaatt gctttctccc aaaaaaagca caatataaag 3600
aaacacaaga tttaattatt tttctacttg gggggaaaaa agtcctcatg tagaagcacc 3660
cacttttgca atgttgttct aagctatcta tctaactctc agcccatgat aaagttcctt 3720
aagctggtga ttcctaatca aggacaagcc accctagtgt ctcatgtttg tatttggtcc 3780
cagttgggta cattttaaaa tcctgatttt ggagacttaa aaccaggtta atggctaaga 3840
atgggtaaca tgactcttgt tggattgtta ttttttgttt gcaatgggga atttataaga 3900
agcatcaagt ctctttctta ccaaagtctt gttaggtggt ttatagttct tttggctaac 3960
aaatcatttt ggaaataaag attttttact acaaaaatg 3999
<210> 178
<211> 1069
<212> DNA
<213> Homo Sapiens
<400> 178
aaaaaagatg aataaatgaa taagagagat gaataaacaa atttacatta catgtgatag 60
ttatcatggt atggccttca tgacaagatg gatgagaata tcactgatag gatattagcc 120
ttctttcata tctttatatt gaaatatggg ctttacttca atttgaaggt ctttcatgaa 180
caataaaaga gagtagaagg actgtctgag aaggcaggag acatataaaa cagatgactg 240
aaagactgac tagctcctgg aaagggaaac atttggaaca tccagagtaa gggcaaatgg 300
gcttctacca gcacaacaaa gagcctccag gtggcaacat ggaagcaggt tatcagagaa 360
aataaatgtg caaattcctt atttacaatg actcacttaa ccccacaaac atgtttcact 420
gctgccttcc ccagttgtcg cttatgtact gttgttacct ttcagttaca tgcctttgat 480
cctaaaattc tctacttttg gtgccttatc agttctttgc aatctgcctg tggttatcag 540
cacttaaagc acaattttga aggggaaaaa aatgataatc accttagtcc caaagaaata 600
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
61
atttgtcaaa ctgccttatt agtattaaaa acagacacac tgaatgaagt agcatgatac 660
gcatatatcc tactcagtat cattggcctt ttatcaaatg gggaaactat acttttgtat 720
tacatagttt tagaaatcga aagttagaga ctctttataa gtaatgtcaa ggaacagtaa 780
tttaaaaaca aagttctaac aaatatattg tttgcttaat cacaatgccc tcaacttgta 840
tttgaataac taaataggac atgtcttcct tggagctgtg ggcattagtt cagaagcact 900
acctgcatct taattttcaa aacttaagtt ttattagcaa atcctcttct ctgtaagact 960
tagctatgaa gtggtatatt ttttccaaat atttttctga aaacatttgt tgttgtaact 1020
gcacaataaa agtccagttg caattaaaaa aaaaaaaaaa aaaaaaaaa 1069
<210> 179
<211> 1817
<212> DNA
<213> Homo Sapiens
<400> 179
tgctattctg ccaaaagaca atttctagag tagttttgaa tgggttgatt tcccccactc 60
ccacaaactc tgaagccagt gtctagctta ctaaaaaaag agttgtatat aatatttaag 120
atgctgagta tttcatagga aagctgaatg ctgctgtaaa gtgctcttta agtctttttt 180
ttttttaatc cccttctaat gaatgaaact aggggaattt caggggacag agatgggatt 240
tgttgtatga taaactgtat gtagttttta gtctttctgt tttgagaagc agtggttggg 300
gcatttttaa gatggctggc tactcttgtt ttccctcatg ataataaatt tgtcataact 360
cagtaacatg aacttgcccc tagaggtagt tgttaataat tttgaaatat taaggtcttg 420
ccaagcttct gatgattcac acctgtacta ctgattatta agcaggacag actgagcttt 480
ctgttgcaaa taccttggag gagaaagtaa tttctaaata tacagagagg taacttgact 540
atatatgttg catcctgtgc ctcccttcat attaatattt gataaagatt ttaatttatg 600
taaaacttct aaagcagaat caaagctcct cttggggaaa tggcaagtct ttaggatagg 660
caagaccctg tatgaatagt accaaagcat taccgcatgg tagagaacac actcgattaa 720
aaatgttaag ctatctgaaa aataaaatgt gcaagtcttc aggatggcac aaaacaaagg 780
ttaatgcttc ttggggcaca tttcttagag ggcttgctga gtgtgtaaat ataatcgact 840
tttgtttgtg ttacatgact tctgtgactt cattgaaaat ctgcacaatt cagtttcagc 900
tctggattac ttcagttgac ctttgtgaag gtttttatct gtgtagaatg ggtgtttgac 960
ttgttttagc ctattaaatt tttattttct ttcactctgt attaaaagta aaacttacta 1020
aaagaaaaga ggtttgtgtt cacattaaat ggttttggtt tggcttcttt tagtcaggct 1080
ttctgaacat tgagatatcc tgaacttaga gctcttcaat cctaagattt tcatgaaaag 1140
cctctcactt gaacccaaac cagagtactc ttactgcctc ttttctaaat gttcaggaaa 1200
agcattgcca gttcagtctt ttcaaaatga gggagaaaca tttgcctgcc ttgtaataac 1260
aagactcagt gcttattttt taaactgcat tttaaaaatt ggatagtata ataacaataa 1320
ggagtaagcc accttttata ggcaccctgt agttttatag ttcttaatct aaacatttta 1380
tatttccttc ttttggaaaa aacctacatg ctacaagcca ccatatgcac agactataca 1440
gtgagttgag ttggctctcc cacagtcttt gaggtgaatt acaaaagtcc agccattatc 1500
atcctcctga gttatttgaa atgatttttt ttgtacattt tggctgcagt attggtggta 1560
gaatatacta taatatggat catctctact tctgtattta tttatttatt actagacctc 1620
aaccacagtc ttctttttcc ccttccacct ctctttgcct gtaggatgta ctgtatgtag 1680
tcatgcactt tgtattaata tattagaaat ctacagatct gttttgtact ttttatactg 1740
ttggatactt ataatcaaaa cttttactag ggtattgaat aaatctagtc ttactagaaa 1800
aaaaaaaaaa aaaaaaa 1817
<210> 180
<211> 2382
<212> DNA
<213> Homo Sapiens
<400> 180
acttttattg gaagcagcag ccacatccct gcatgatttg cattgcaata caaccataac 60
cgggcagcca ctcctgagtg ataaccagta taacataaac gtagcagcct caatttttgc 120
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
62
ctttatgacg acagcttgtt atggttgcag tttgggtctg gctttacgaa gatggcgacc 180
gtaacactcc ttagaaactg gcagtcgtat gttagtttca cttgtctact ttatatgtct 240
gatcaatttg gataccattt tgtccagatg caaaaacatt ccaaaagtaa tgtgtttagt 300
agagagagac tctaagctca agttctggtt tatttcatgg atggaatgtt aattttatta 360
tgatattaaa gaaatggcct tttattttac atctctcccc tttttccctt tcccccttta 420
ttttcctcct tttctttctg aaagtttcct tttatgtcca taaaatacaa atatattgtt 480
cataaaaaat tagtatccct tttgtttggt tgctgagtca cctgaacctt aattttaatt 540
ggtaattaca gcccctaaaa aaaacacatt tcaaataggc ttcccactaa actctatatt 600
ttagtgtaaa ccaggaattg gcacactttt tttagaatgg gccagatggt aaatatttat 660
gcttcacggt ccatacagtc tctgtcacaa ctattcagtt ctgctagtat agcgtgaaag 720
cagctataca caatacagaa atgaatgagt gtggttatgt tctaataaaa cttatttata 780
aaaacaaggg gaggctgggt ttagcctgtg ggccatagtt tgtcaaccac tggtgtaaaa 840
ccttagttat atatgatctg cattttcttg aactgatcat tgaaaactta taaacctaac 900
agaaaagcca cataatattt agtgtcatta tgcaataatc acattgcctt tgtgttaata 960
gtcaaatact tacctttgga gaatacttac ctttggagga atgtataaaa tttctcaggc 1020
agagtcctgg atataggaaa aagtaattta tgaagtaaac ttcagttgct taatcaaact 1080
aatgatagtc taacaactga gcaagatcct. catctgagag tgcttaaaat gggatcccca 1140
gagaccatta accaatactg gaactggtat ctagctactg atgtcttact ttgagtttat 1200
ttatgcttca gaatacagtt gtttgccctg tgcatgaata tacccatatt tgtgtgtgga 1260
tatgtgaagc ttttccaaat agagctctca gaagaattaa gtttttactt ctaattattt 1320
tgcattactt tgagttaaat ttgaatagag tattaaatat aaagttgtag attcttatgt 1380
gtttttgtat tagcccagac atctgtaatg tttttgcact ggtgacagac aaaatctgtt 1440
ttaaaatcat atccagcaca aaaactattt ctggctgaat agcacagaaa agtattttaa 1500
cctacctgta gagatcctcg tcatggaaag gtgccaaact gttttgaatg gaaggacaag 1560
taagagtgag gccacagttc ccaccacacg agggcttttg tattgttcta ctttttcagc 1620
cctttacttt ctggctgaag catccccttg gagtgccatg tataagttgg gctattagag 1680
ttcatggaac atagaacaac catgaatgag tggcatgatc cgtgcttaat gatcaagtgt 1740
tacttatcta ataatcctct agaaagaacc ctgttagatc ttggtttgtg ataaaaatat 1800
aaagacagaa gacatgagga aaaacaaaag gtttgaggaa atcaggcata tgactttata 1860
cttaacatca gatcttttct ataatatcct actactttgg ttttcctagc tccataccac 1920
acacctaaac ctgtattatg aattacatat tacaaagtca taaatgtgcc atatggatat 1980
acagtacatt ctagttggaa tcgtttactc tgctagaatt taggtgtgag attttttgtt 2040
tcccaggtat agcaggctta tgtttggtgg cattaaattg gtttctttaa aatgctttgg 2100
tggcactttt gtaaacagat tgcttctaga ttgttacaaa ccaagcctaa gacacatctg 2160
tgaatactta gatttgtagc ttaatcacat tctagacttg tgagttgaat gacaaagcag 2220
ttgaacaaaa attatggcat ttaagaattt aacatgtctt agctgtaaaa atgagaaagt 2280
gttggttggt tttaaaatct ggtaactcca tgatgaaaag aaatttattt tatacgtgtt 2340
atgtctctaa taaagtattc atttgataaa aaaaaaaaaa as 2382
<210> 181
<211> 2377
<212> DNA
<213> Homo Sapiens
<400> 181
atctttatgc aagacaagag tcagccatca gacactgaaa tatattatga tagattatga 60
agaattttct ctgtagaatt atattcttcc tggaacctgg tagagtagat tagactcaaa 120
ggctttttct tccttttctt actcctgttt tttccactca ctcttcccaa gagatttcct 180
aaagcttcaa gcttaataag cctaatagtg aaaaataact gaatttaatg gtataatgaa 240
gttcttcatt tccagacatc tttaattgat cttaaagctc atttgagtct ttgcccctga 300
acaaagacag acccattaaa atctaagaat tctaaatttt cacaactgtt tgagcttctt 360
ttcattttga aggatttgga atatatatgt tttcataaaa gtatcaagtg aaatatagtt 420
acatgggagc tcaatcatgt gcagattgca ttctgttatg ttgactcaat atttaattta 480
caactatcct tatttatatt gacctcaaga actccatttt atgcaatgca gaccactgag 540
atatagctaa cattctttca aataattttc cttttctttt ataattcctc tatagcaaat 600
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
63
ttttatgtat aactgattat acatatccat atttatattt cattgattcc aagacatcac 660
tttttcaatt taacatctct gaaattgtga catttcttgc aactgttggc acttcagatg 720
cagtgtttaa aattatgctt gaataaatat tacactaatc caactttacc taaatgttta 780
tgcatctagg caaattttgt tttcttataa agatttgaga gcccatttat gacaaaatat 840
gaaggcgaaa tttaaggaca actgagtcac gcacaactca acatggagcc taactgatta 900
tcagctcaga tcccgcatat cttgagttta caaaagctct ttcaggtccc catttatact 960
ttacgtgagt gcgaatgatt tcagcaaacc ctaacttaac taacaagaat gggtaggtat 1020
gtctacgttt cattaacaaa tttttattat ttttattcta ttatatgaga tccttttata 1080
ttatcatctc acttttaaac aaaattaact ggaaaaatat tacatggaac tgtcatagtt 1140
aggttttgca gcatcttaca tgtcttgtat caatggcagg agaaaaatat gataaaaaca 1200
atcagtgctg tgaaaaacaa ctttcttcta gagtcctctt actttttatt cttctttatc 1260
atttgtgggt ttttccccct tggctctcac tttaacttca agcttatgta acgactgtta 1320
taaaactgca tatttaaatt atttgaatta tatgaaataa ttgttcagct atctgggcag 1380
ctgttaatgt aaacctgaga gtaataacac tactctttta tctacctgga atacttttct 1440
gcataaaatt tatctttgta agctaactct attaatcagg tttcttctag cctctgcaac 1500
ctacttcagt tagaattgtc taatactgct ctattaatca ggtttctacc ctctacaacc 1560
tacttcagtt aaaattgtct aatacagcaa tatttaaaaa aaaaacactg caattgtcaa 1620
ggatggaaaa tgtgtgattt gtgtaaacaa tttttaccaa ctttacattt tcctacagat 1680
aaatgtgaaa ttttgataag aagtctacgc aatgacaagt acggtacata aattttatta 1740
agaatattga gtataaagta ctttaattct aaattataag aaaatataca tttgcacata 1800
ttaatataga aattcatttt gtgtatattt aacatagctt ttaaactatt ttacattagc 1860
tacttcatta tggtttcttg aacttctgaa aaaaattaga aatgtattaa acttatcagt 1920
aacataaaaa cttattttgt ttcacctaac gaatactgcg tttgtaaaaa taaatttaat 1980
atagaatata tttttaaatt aaatatttga atataaaata gctctaagaa agaagcaaat 2040
tatcactgaa catatttctt attatttctg gctttgaatt atacgtaact taaattgtct 2100
taaatgatac agaatattgg agaatatgat actttcacat aatatactat gaacctgttc 2160
atataactct gattgactac taacttctgt tttatgtatt tattaaagag ctgacactgt 2220
agtttgtggt gagatgttta tttttctaac agagcttata acagttagga caaggcattt 2280
aattaatgca tcattctgtt tagtagtagg tgttaatcaa tatgaaattc tctgttttaa 2340
aataaaaatg taaaaatcta aaaaaaaaaa aaaaaaa 2377
<210> 182
<211> 1370
<212> DNA
<213> Homo Sapiens
<400> 182
tgtgagcatg gtattttgtc tcggaagaaa aaaatatggg tcaggcgcaa agtaagccca 60
ccccactggg aactatgtta aaaaaaaatt tcaagattta agggagatta cggtgttact 120
atgacaccag aaaaacttag aactttgtgt gaaatagact ggctaacatt agaggtgggt 180
tggctatcag aagaaagcct ggagaggtcc cttgtttcaa aggtatggca caaggtaacc 240
tgtaagccaa agcacccgga ccagtttcta tacatagaca gttacagctg gtttagaccc 300
cttccccctc tccccacagt agttaagaga acagcagcat aagcagctgg cagaggcaag 360
gaaagaccag cagagagaaa aaaaggccat ctataccaat tttaagttaa tttagactga 420
acaagggctt attaatagca aaggataatt gaaatcacaa acttataagg gtttcaacaa 480
aagtgaagtt tgctaaaagt taacagtgta acatgtatta tggtaacttc taatcttgtg 540
gccttagaca gtctagtcaa aacacataaa gaaagtttgc tttaaaaaaa caatggttat 600
cttcaaaaat aaaggggaga ggcagaattt atataaaaag agttatatga taaattcttg 660
tcctgaaata aattaactgg ttgtttaaag aaaagaatgt ttgtaataag tcaaaaagtt 720
aaaacatgtt taaaaaattg tctgcaaaag tcataaaaga aaaaatttta ttaaaaaaat 780
tttaagcaaa aaatgttgta taatttaaaa gtaataaggc ctcctgtgta ctattaagac 840
agatgcaaat tcctggttga aatggatcaa atattccatc tgcacattaa acaaaagcaa 900
ttgttatgct tgtgcacatg gcaggccaga ggccctgatt gtcccccttc cactaaggtg 960
gtcctctagt cgaccaggcg tggactgcat ggtagctctt ttccaggatt ctacagcctg 1020
gagtaataag tcatgccaag ctctctctgc tatatcccaa agtctctgcg ggtcagcccc 1080
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
64
caagggccat gcagcttctg tctcccaaca ctaagttcac ttcgtgtctc tcacggcaga 1140
gaggaaactt agtattcctt ggagacctga agggatgcag tgagcttaag aattttcaag 1200
agcttatcaa tcagtcagcc cttgttcatc cccgagtgga tgtgtggtgg tattgtggtg 1260
gacctttact gggcactctg ccaaataact agtgtggcac ttgtgcttta gtccatttgg 1320
ctatcccttt caccctggca tttcatcaac caaaaaaaaa aaaaaaaaaa 1370
<210> 183
<211> 2060
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<222> (1). .(2060)
<223> n=A,T,C or G
<400> 183
gtttcagggg aggagacaag gtttcttgtt tgccgtatat gctcctgcag agaagaggaa 60
gtgaccgtgg aggccatctg gccctgtgtt ttgatatggc aaaattaatg aatgcaatca 120
gaagaccttt gagcaagaaa gtaccctgga acaacccaat ttggactgca agtattagtt 180
gggtcttcca ggtgcctctc acagcagcag tcatggcagc agtgactcta gccatgtcca 240
tgaccaactg ctgcataaca aatagccccg agactcagca gcttacaaca gggtccccag 300
cccacagact ggcactggtc catggcttgt taggaacctg actgcgcagc agaaggtgag 360
tgagcattac tgcctgagct ctgcctcctg tcagatcatc aggggcatta gattctcata 420
ggagcgtgaa ccctattgca aaccgcgcat gcgaaggatg tacgttgcgt gctccttatg 480
agaatctaac taatgcctga tgatttgagg tggggcagtt tcatccccaa accatctctc 540
tcccttcatg tccatggaaa aattgtcttc tacaaaacca gtccgtggtg ccaaaaaggt 600
tggagactgc tggtttacaa ccgcaatgaa cattcatcat cccacacagt gtcagagggt 660
cgggaacacg ggtgccctgc ctgtgtgctt ccggttccag atttctcagt gggttgtgat 720
caaggtatca gcggaggccg tattcatctg caagcttgac caggaataga agagccactt 780
catgggtggc tcactcagat gccagcaggt cagtgctggt ggctggcagg cagcctcagc 840
tcctcacctc atggatctct cctgagcaca gttttcctgt ccttacaacc tggtagctgg 900
cttctccaga gcaggtgact caggagagga caaggtgaga gcccagcacc ttatggtcta 960
gtctcagaag tcacacgcca tcatttctgc aatgtcattt tggggttcca ggtcagctgt 1020
atcactgtgg gaggtgagta tatagatgtc ctagaccatt caggctgcta tgacagaaca 1080
ccatgaactg agtggctcat gaacaacaga aatttcccac agttctgtag gctgggaaat 1140
ccaagatcaa ggtggcagca ggttcagcgt ctgctaagct cctgcttttc atggattgca 1200
tcttctcact gtgtcctcac gtgatggaca gagcaaatga gctctcaggc actagtccca 1260
gccatgagga ctctgctttc atgactcatc actccgcaaa ggcccacctc catcagaaga 1320
cagctgctaa ctgcagctgc catcctccaa gacgggagac acagaattgg gggacatata 1380
cattgagatc tgaaaggcct ggacagcaac aggtggggat cgtgggggca tcttggaggg 1440
tggctgccgc agtaacattt ctgacccatg ctttctgctt gcactcatct cctgcctttg 1500
atcttcatta tctcargcag tccccacaac gactgtatct aggagttcat tttaccctca 1560
ttttacagat gaaacgtctc agagggtaat gtgcttgccc agtgtctcac aaatgcaaag 1620
tcactgaggt aggatttcaa cctaggtcca atcatctctg cagcattagg ggttcaccat 1680
tgccatagac ttaactgtgt cccccaaaat ttgtatgttg aagccctacc agcctccccc 1740
ccccaatgtg ctgatgtttg gagaaagggc ctttgggagg taattaggtt tagatgagat 1800
catgagggtg ggactctcat aatggcatta atgccatcag gtgaagagat accagagacc 1860
ttgtgtcctc tctctctgca atgtgaggac acagtgagaa ggcagctgtc tgcaagctgg 1920
gaagagagta ctgaccagga acttaatcag agggcatctt gatcttggac ttcccagcct 1980
ccagaactct gaaaagttaa tgnctattat ttaagccacg cagtctatgg aattttgtta 2040
gagccaaccc caagcttact 2060
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
<210> 184
<211> 3079
<212> DNA
<213> Homo sapiens
<400> 184
ggcacaaagt tgggggccgc gaagatgagg ctgtccccgg cgcccctgaa gctgagccgg 60
actccggcac tgctggccct ggcgctgccc ctggccgcgg cgctggcctt ctccgacgag 120
accctggaca aagtgcccaa gtcagagggc tactgtagcc gtatcctgcg cgcccagggc 180
acgcggcgcg agggctacac cgagttcagc ctccgcgtgg agggcgaccc cgacttctac 240
aagccgggaa ccagctaccg cgtaacactt tcagctgctc ctccctccta cttcagagga 300
ttcacattaa ttgccctcag agagaacaga gagggtgata aggaagaaga ccatgctggg 360
accttccaga tcatagacga agaagaaact cagtttatga gcaattgccc tgttgcagtc 420
actgaaagca ctccacggag gaggacccgg atccaggtgt tttggatagc accaccagcg 480
ggaacaggct gcgtgattct gaaggccagc atcgtacaaa aacgcattat ttattttcaa 540
gatgagggct ctctgaccaa gaaactttgt gaacaagatt ccacatttga tggggtgact 600
gacaaaccca tcttagactg ctgtgcctgc ggaactgcca agtacagact cacattttat 660
gggaattggt ccgagaagac acacccaaag gattaccctc gtcgggccaa ccactggtct 720
gcgatcatcg gaggatccca ctccaagaat tatgtactgt gggaatatgg aggatatgcc 780
agcgaaggcg tcaaacaagt tgcagaattg ggctcacccg tgaaaatgga ggaagaaatt 840
cgacaacaga gtgatgaggt cctcaccgtc atcaaagcca aagcccaatg gccagcctgg 900
cagcctctca acgtgagagc agcaccttca gctgaatttt ccgtggacag aacgcgccat 960
ttaatgtcct tcctgaccat gatgggccct agtcccgact ggaacgtagg cttatctgca 1020
gaagatctgt gcaccaagga atgtggctgg gtccagaagg tggtgcaaga cctgattccc 1080
tgggacgctg gcaccgacag cggggtgacc tatgagtcac ccaacaaacc caccattccc 1140
caggagaaaa tccggcccct gaccagcctg gaccatcctc agagtccttt ctatgaccca 1200
gagggtgggt ccatcactca agtagccaga gttgtcatcg agagaatcgc acggaagggt 1260
gaacaatgca atattgtacc tgacaatgtc gatgatattg tagctgacct ggctccagaa 1320
gagaaagatg aagatgacac ccctgaaacc tgcatctact ccaactggtc cccatggtcc 1380
gcctgcagct cctccacctg tgacaaaggc aagaggatgc gacagcgcat gctgaaagca 1440
cagctggacc tcagcgtccc ctgccctgac acccaggact tccagccctg catgggccct 1500
ggctgcagtg acgaagacgg ctccacctgc accatgtccg agtggatcac ctggtcgccc 1560
tgcagcatct cctgcggcat gggcatgagg tcccgggaga ggtatgtgaa gcagttcccg 1620
gaggacggct ccgtgtgcac gctgcccact gaggaaatgg agaagtgcac ggtcaacgag 1680
gagtgctctc ccagcagctg cctgatgacc gagtggggcg agtgggacga gtgcagcgcc 1740
acctgcggca tgggcatgaa gaagcggcac cgcatgatca agatgaaccc cgcagatggc 1800
tccatgtgca aagccgagac atcacaggca gagaagtgca tgatgccaga gtgccacacc 1860
atcccatgct tgctgtcccc atggtccgag tggagtgact gcagcgtgac ctgcgggaag 1920
ggcatgcgaa cccgacagcg gatgctcaag tctctggcag aacttggaga ctgcaatgag 1980
gatctggagc aggtggagaa gtgcatgctc cctgaatgcc ccattgactg tgagctcacc 2040
gagtggtccc agtggtcgga atgtaacaag tcatgtggga aaggccacgt gattcgaacc 2100
cggatgatcc aaatggagcc tcagtttgga ggtgcaccct gcccagagac tgtgcagcga 2160
aaaaagtgcc gcatccgaaa atgccttcga aatccatcca tccaaaagcc acgctggagg 2220
gaggcccgag agagccggcg gagtgagcag ctgaaggaag agtctgaagg ggagcagttc 2280
ccaggttgta ggatgcgccc atggacggcc tggtcagaat gcaccaaact gtgcggaggt 2340
ggaattcagg aacgttacat gactgtaaag aagagattca aaagctccca gtttaccagc 2400
tgcaaagaca agaaggagat cagagcatgc aatgttcatc cttgttagca agggtacgag 2460
ttccccaggg ctgcactcta gattccagag tcaccaatgg ctggattatt tgcttgttta 2520
agacaattta aattgtgtac gctagttttc atttttgcag tgtggttcgc ccagtagtct 2580
tgtggatgcc agagacatcc tttctgaata cttcttgatg ggtacaggct gagtggggcg 2640
ccctcacctc cagccagcct cttcctgcag aggagtagtg tcagccacct tgtactaagc 2700
tgaaacatgt ccctctggag cttccacctg gccagggagg acggagactt tgacctactc 2760
cacatggaga ggcaaccatg tctggaagtg actatgcctg agtcccaggg tgcggcaggt 2820
aggaaacatt cacagatgaa gacagcagat tccccacatt ctcatctttg gcctgttcaa 2880
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
66
tgaaaccatt gtttgcccat ctcttcttag tggaacttta ggtctctttt caagtctcct 2940
cagtcatcaa tagttcctgg ggaaaaacag agctggtaga cttgaagagg agcattgatg 3000
ttgggtggct tttgttcttt cactgagaaa ttcggaatac atttgtctca cccctgatat 3060
tggttcctga tgccccagc 3079
<210> 185
<211> 3000
<212> DNA
<213> Homo Sapiens
<400> 185
gtttcagggg aggagacaag gtttcttgtt tgccgtatat gctcctgcag agaagaggaa 60
gtgaccgtgg aggccatctg gccctgtgtt ttgatatggc aaaattaatg aatgcaatca 120
gaagaccttt gagcaagaaa gtaccctgga acaacccaat ttggactgca agtattagtt 180
gggtcttcca ggtgcctctc acagcagcag tcatggcagc agtgactcta gccatgtcca 240
tgaccaactg ctgcataaca aatagccccg agactcagca gcttacaaca gggtccccag 300
cccacagact ggcactggtc catggcttgt taggaacctg actgcgcagc agaaggtgag 360
tgagcattac tgcctgagct ctgcctcctg tcagatcatc aggggcatta gattctcata 420
ggagcgtgaa ccctattgca aaccgcgcat gcgaaggatg tacgttgcgt gctccttatg 480
agaatctaac taatgcctga tgatttgagg tggggcagtt tcatccccaa accatctctc 540
tcccttcatg tccatggaaa aattgtcttc tacaaaacca gtccgtggtg ccaaaaaggt 600
tggagactgc tggtttacaa ccgcaatgaa cattcatcat cccacacagt gtcagagggt 660
cgggaacacg ggtgccctgc ctgtgtgctt ccggttccag atttctcagt gggttgtgat 720
caaggtatca gcggaggccg tattcatctg caagcttgac caggaataga agagccactt 780
catgggtggc tcactcagat gccagcaggt cagtgctggt ggctggcagg cagcctcagc 840
tcctcacctc atggatctct cctgagcaca gttttcctgt ccttacaacc tggtagctgg 900
cttctccaga gcaggtgact caggagagga caaggtgaga gccacagcac cttatggtct 960
agtctcagaa gtcacacgcc atcatttctg caatgtcatt ttggggttcc aggtcagctg 1020
tatcactgtg ggaggtgagt atatagatgt cctagaccat tcaggctgct atgacagaac 1080
accatgaact gagtggctca tgaacaacag aaatttccca cagttctgta ggctgggaaa 1140
tccaagatca aggtggcagc aggttcagcg tctgctaagc tcctgctttt catggattgc 1200
atcttctcac tgtgtcctca cgtgatggac agagcaaatg agctctcagg cactagtccc 1260
agccatgagg actctgcttt catgactcat cactccgcaa aggcccacct ccatcagaag 1320
acagctgcta actgcagctg ccatcctcca agacgggaga cacagaattg ggggacatat 1380
acattgagat ctgaaaggcc tggacagcaa caggtgggga tcgtgggggc atcttggagg 1440
gtggctgccg cagtaacatt tctgacccat gctttctgct tgcactcatc tcctgccttt 1500
gatcttcatt atctcaggca gtccccacaa cgactgtatc taggagttca ttttaccctc 1560
attttacaga tgaaacgtct cagagggtaa tgtgcttgcc cagtgtctca caaatgcaaa 1620
gtcactgagg taggatttca acctaggtcc aatcatctct gcagcattag gggttcacca 1680
ttgccataga cttaactgtg tcccccaaaa tttgtatgtt gaagccctac cagcctcccc 1740
cccccaatgt gctgatgttt ggagaaaggg cctttgggag gtaattaggt ttagatgaga 1800
tcatgagggt gggactctca taatggcatt aatgccatca ggtgaagaga taccagagac 1860
cttgtgtcct ctctctctgc aatgtgagga cacagtgaga aggcagctgt ctgcaagctg 1920
ggaagagagt actgaccagg aacttaatca gagggcatct tgatcttgga cttcccagcc 1980
tccagaactc tgaaaagtta atgtctatta tttaagccac gcagtctatg gaattttgtt 2040
agagccaacc caagcttact aagataatca gtatgctgca ctttctataa atgtaatttt 2100
tacatttata aaaacaaaac aagagatttg ctgctctata acaactgtac ctacattgta 2160
gatggaataa caaatctaca tacagattta gtaatctcta tgtagatata gaacatagtg 2220
tatctaatag agacatagtg tctgtggtct gatgttaatt ttaggaatta gccgtcactg 2280
attgggcctt gtccaggtat tcttctccct tgtcctggct ctgtaaccta gttatccttg 2340
tctttgctaa cccataacca actattgtat caggactatt atgccactac agatgatgca 2400
gtttgggttt actgtttctc accatttaga caatacttca tcaaatatat ttctgtatga 2460
ctttagtgat atcagttttt gattcattcc tgcatagatc tgggcaaatt gtagacctta 2520
ggaggtgtat tcaccatcca gttctctgga actgcttatg acatttttct ctgagctttc 2580
ttgtcccaaa aggagccttc ctaaaatagt ctttaagtgc ctttaaaaag agaaagagaa 2640
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
67
attaagagaa aaaaaacccc aaactcattc ctttactctg atgtgacagt cctcccagga 2700
cactgcagtg gcctgagttt tgctgttaat ttcattcact tatgtttggg ctatgtaaat 2760
tctgcctaga gctggaatgt cattatgtaa agaaatattt tttgtttata ttctttaata 2820
gtaccagtaa tgtatatctt attcagcttc gagaatataa ttgggttgtt tataaaaacc 2880
acacatcatc aaactcacat tgtaacgatt atttcacttt tcaaaaaaaa tggcattaga 2940
aaaacttgaa tgatgttagt tatcttaaag aagtgtgtac tatgtttaaa aaaaaaaaaa 3000
<210> 186
<211> 807
<212> PRT
<213> Homo Sapiens
<400> 186
Met Arg Leu Ser Pro Ala Pro Leu Lys Leu Ser Arg Thr Pro Ala Leu
10 15
Leu Ala Leu Ala Leu Pro Leu Ala Ala Ala Leu Ala Phe Ser Asp Glu
20 25 30
Thr Leu Asp Lys Val Pro Lys Ser Glu Gly Tyr Cys Ser Arg Ile Leu
35 40 45
Arg Ala Gln Gly Thr Arg Arg Glu Gly Tyr Thr Glu Phe Ser Leu Arg
50 55 60
Val Glu Gly Asp Pro Asp Phe Tyr Lys Pro Gly Thr Ser Tyr Arg Val
65 70 75 80
Thr Leu Ser Ala Ala Pro Pro Ser Tyr Phe Arg Gly Phe Thr Leu Ile
85 90 95
Ala Leu Arg Glu Asn Arg Glu Gly Asp Lys Glu Glu Asp His Ala Gly
100 105 110
Thr Phe Gln Ile Ile Asp Glu Glu Glu Thr Gln Phe Met Ser Asn Cys
115 120 125
Pro Val Ala Val Thr Glu Ser Thr Pro Arg Arg Arg Thr Arg Ile Gln
130 135 140
Val Phe Trp Ile Ala Pro Pro Ala Gly Thr Gly Cys Val Ile Leu Lys
145 150 155 160
Ala Ser Ile Val Gln Lys Arg Ile Ile Tyr Phe Gln Asp Glu Gly Ser
165 170 175
Leu Thr Lys Lys Leu Cys Glu Gln Asp Ser Thr Phe Asp Gly Val Thr
180 185 190
Asp Lys Pro Ile Leu Asp Cys Cys Ala Cys Gly Thr Ala Lys Tyr Arg
195 200 205
Leu Thr Phe Tyr Gly Asn Trp Ser Glu Lys Thr His Pro Lys Asp Tyr
210 215 220
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
68
Pro Arg Arg Ala Asn His Trp Ser Ala Ile Ile Gly Gly Ser His Ser
225 230 235 240
Lys Asn Tyr Val Leu Trp Glu Tyr Gly Gly Tyr Ala Ser Glu Gly Val
245 250 255
Lys Gln Val Ala Glu Leu Gly Ser Pro Val Lys Met Glu Glu Glu Ile
260 265 270
Arg Gln Gln Ser Asp Glu Val Leu Thr Val Ile Lys Ala Lys Ala Gln
275 280 285
Trp Pro Ala Trp Gln Pro Leu Asn Val Arg Ala Ala Pro Ser Ala Glu
290 295 300
Phe Ser Val Asp Arg Thr Arg His Leu Met Ser Phe Leu Thr Met Met
305 310 315 320
Gly Pro Ser Pro Asp Trp Asn Val Gly Leu Ser Ala Glu Asp Leu Cys
325 330 335
Thr Lys Glu Cys Gly Trp Val Gln Lys Val Val Gln Asp Leu Ile Pro
340 345 350
Trp Asp Ala Gly Thr Asp Ser Gly Val Thr Tyr Glu Ser Pro Asn Lys
355 360 365
Pro Thr Ile Pro Gln Glu Lys Ile Arg Pro Leu Thr Ser Leu Asp His
370 375 380
Pro Gln Ser Pro Phe Tyr Asp Pro Glu Gly Gly Ser Ile Thr Gln Val
385 390 395 400
Ala Arg Val Val Ile Glu Arg Ile Ala Arg Lys Gly Glu Gln Cys Asn
405 410 415
Ile Val Pro Asp Asn Val Asp Asp Ile Val Ala Asp Leu Ala Pro Glu
420 425 430
Glu Lys Asp Glu Asp Asp Thr Pro Glu Thr Cys Ile Tyr Ser Asn Trp
435 440 445
Ser Pro Trp Ser Ala Cys Ser Ser Ser Thr Cys Asp Lys Gly Lys Arg
450 455 460
Met Arg Gln Arg Met Leu Lys Ala Gln Leu Asp Leu Ser Val Pro Cys
465 470 475 480
Pro Asp Thr Gln Asp Phe Gln Pro Cys Met Gly Pro Gly Cys Ser Asp
485 490 495
Glu Asp Gly Ser Thr Cys Thr Met Ser Glu Trp Ile Thr Trp Ser Pro
500 505 510
Cys Ser Ile Ser Cys Gly Met Gly Met Arg Ser Arg Glu Arg Tyr Val
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
69
515 520 525
Lys Gln Phe Pro Glu Asp Gly Ser Val Cys Thr Leu Pro Thr Glu Glu
530 535 540
Met Glu Lys Cys Thr Val Asn Glu Glu Cys Ser Pro Ser Ser Cys Leu
545 550 555 560
Met Thr Glu Trp Gly Glu Trp Asp Glu Cys Ser Ala Thr Cys Gly Met
565 570 575
Gly Met Lys Lys Arg His Arg Met Ile Lys Met Asn Pro Ala Asp Gly
580 585 590
Ser Met Cys Lys Ala Glu Thr Ser Gln Ala Glu Lys Cys Met Met Pro
595 600 605
Glu Cys His Thr Ile Pro Cys Leu Leu Ser Pro Trp Ser Glu Trp Ser
610 615 620
Asp Cys Ser Val Thr Cys Gly Lys Gly Met Arg Thr Arg Gln Arg Met
625 630 635 640
Leu Lys Ser Leu Ala Glu Leu Gly Asp Cys Asn Glu Asp Leu Glu Gln
645 650 655
Val Glu Lys Cys Met Leu Pro Glu Cys Pro Ile Asp Cys Glu Leu Thr
660 665 670
Glu Trp Ser Gln Trp Ser Glu Cys Asn Lys Ser Cys Gly Lys Gly His
675 680 685
Val Ile Arg Thr Arg Met Ile Gln Met Glu Pro Gln Phe Gly Gly Ala
690 695 700
Pro Cys Pro Glu Thr Val Gln Arg Lys Lys Cys Arg Ile Arg Lys Cys
705 710 715 720
Leu Arg Asn Pro Ser Ile Gln Lys Pro Arg Trp Arg Glu Ala Arg Glu
725 730 735
Ser Arg Arg Ser Glu Gln Leu Lys Glu Glu Ser Glu Gly Glu Gln Phe
740 745 750
Pro Gly Cys Arg Met Arg Pro Trp Thr Ala Trp Ser Glu Cys Thr Lys
755 760 765
Leu Cys Gly Gly Gly Ile Gln Glu Arg Tyr Met Thr Val Lys Lys Arg
770 775 780
Phe Lys Ser Ser Gln Phe Thr Ser Cys Lys Asp Lys Lys Glu Ile Arg
785 790 795 800
Ala Cys Asn Val His Pro Cys
805
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
<210> 187
<211> 892
<212> DNA
<213> Homo sapiens
<400> 187
tttattgatg tttcaacagg cacttattca aataagttat atatttgaaa acagccatgg 60
taagcatcct tggcttctca cccattcctc atgtggcatg ctttctagac tttaaaatga 120
ggtaccctga atagcactaa gtgctctgta agctcaagga atctgtgcag tgctacaaag 180
cccacaggca gagaaagaac tcctcaagtg cttgtggtca gagactaggt tccatatgag 240
gcacacctat gatgaaggtc ttcacctcca gaaggtgaca ctgttcagag atcctcattt 300
cctggagagt gggagaaaat ccctcctttg ggaaatccct tttcccagca gcagagccca 360
cctcattgct tagtgatcat ttggaaggca ctgagagcct tcaggggctg acagcagaga 420
aatgaaaatg agtacagttc agatggtgga agaagcatgg cagtgacatc ttccatgctc 480
tttttctcag tgtctgcaac tccaaagatc aaggccataa cccaggagac catcaacgga 540
agattagttc tttgtcaagt gaatgaaatc caaaagcacg catgagacca atgaaagttt 600
ccgcctgttg taaaatctat tttcccccaa ggaaagtcct tgcacagaca ccagtgagtg 660
agttctaaaa gatacccttg gaattatcag actcagaaac ttttattttt tttttctgta 720
acagtctcac cagacttctc ataatgctct taatatattg cacttttcta atcaaagtgc 780
gagtttatga gggtaaagct ctactttcct actgcagcct tcagattctc atcattttgc 840
atctattttg tagccaataa aactccgcac tagcaaaaaa aaaaaaaaaa as 892
<210> 188
<211> 1448
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<222> (1). .(1448)
<223> n = A,T,C or G
<400> 188
tgtgactcac atttctttta ctgtgacaca ataatgtgat cctaaaactg gcttatcctt 60
gagtgtttac aactcaaaca actttttgaa tgcagtagtt tttttttttt aaaaacaaac 120
ttttatgtca aatttttttt cttagaagta gtcttcatta ttataaattt gtacaccaaa 180
aggccatggg gaactttgtg caagtacctc atcgctgagc aaatggagct tgctatgttt 240
taatttcaga aaatttcctc atatacgtag tgtgtagaat caagtctttt aataattcat 300
tttttcttca taatatttac tcaaagttaa gcttaaaaat aagttttatc ttaaaatcat 360
atttgaagac agtaagacag taaactattt taggaagtca acccccattg cactctgtgg 420
cagttattct ggtaaaaata ggcaaaagtg acctgaatct acaatggtgt cccaaagtaa 480
ccaagtaaga gagattgtaa atgataaacc gagctttaaa ggataaagtg ttaataaaga 540
aaggaagctg ggcacatgtc aaaaagggag atcgaaatgt taggtaatca tttagaaagg 600
acagaaaata tttaaagtgg ctcataggta atgaatattt ctgacttaga tgtaaatcca 660
tctggaatct ttacatcctt tgccagctga aacaagaaag tgaagggaca atgatatttc 720
atggtcagtt tattttgtaa gagacagaag aaattatatc tatacattac cttgtagcag 780
cagtacctgg aagccccagc ccgtcacaga agtgtggagg ggggctcctg actagacaat 840
ttccctagcc cttgtgattt gaagcatgaa agttctggca ggttatgagc agcactaggg 900
ataaagtatg gttttatttt ggtgtaattt aggtttttca acaaagccct tgtctaaaat 960
aaaaggcatt attggaaata tttgaaaact agaaaatgat ggataaaagg gctgataaga 1020
aaatttctga ctgtcagtag aagtgagata agatcctcag aggaaacagt aagaagggat 1080
aatcattaag atagtaaaac aggcaaagca gaatcacatg tgcncacaca catacacatg 1140
taaacattgg aatgcataag ttttaatatt ttagcgctat cagtttctaa atgcattaat 1200
tactaactgc cctctcccaa gattcattta gttcaaacag tatccgtaaa ctaggaataa 1260
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
71
tgccacatgc attcaatggg atcttttaag tactcttcag tttgttccaa gaaatgtgcc 1320
tactgaaatc aaattaattt gtattcaatg tgtacttcaa gactgctaat tgtttcatct 1380
gaaagcctac aatgaatcat tgttcamcct tgaaaaataa aattttgtaa atcaaaaaaa 1440
aaaaaaaa 1448
<210> 189
<211> 460
<212> DNA
<213> Homo sapiens
<400> 189
ttttgggagc acggactgtc agttctctgg gaagtggtca gcgcatcctg cagggcttct 60
cctcctctgt cttttggaga accagggctc ttctcagggg ctctagggac tgccaggctg 120
tttcagccag gaaggccaaa atcaagagtg agatgtagaa agttgtaaaa tagaaaaagt 180
ggagttggtg aatcggttgt tctttcctca catttggatg attgtcataa ggtttttagc 240
atgttcctcc ttttcttcac cctccccttt tttcttctat taatcaagag aaacttcaaa 300
gttaatggga tggtcggatc tcacaggctg agaactcgtt cacctccaag catttcatga 360
aaaagctgct tcttattaat catacaaact ctcaccatga tgtgaagagt ttcacaaatc 420
cttcaaaata aaaagtaatg acttaaaaaa aaaaaaaaaa 460
<210> 190
<211> 481
<212> DNA
<213> Homo sapiens
<400> 190
aggtggtgga agaaactgtg gcacgaggtg actgaggtat ctgtgggagc taatcctgtc 60
caggtggaag taggagaatt tgatgatggt gcagaggaaa ccgaagagga ggtggtggcg 120
gaaaatccct gccagaacca ccactgcaaa cacggcaagg tgtgcgagct ggatgagaac 180
aacaccccca tgtgcgtgtg ccaggacccc accagctgcc cagcccccat tggcgagttt 240
gagaaggtgt gcagcaatga caacaagacc ttcgactctt cctgccactt ctttgccaca 300
aagtgcaccc tggagggcac caagaagggc cacaagctcc acctggacta catcgggcct 360
tgcaaataca tccccccttg cctggactct gagctgaccg aattccccct gcgcatgcgg 420
gactggctca agaacgtcct ggtcaccctg tatgagaggg atgaggacaa caaccttctg 480
a 481
<210> 191
<211> 489
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<222> (1) . . (489)
<223> n = A,T,C or G
<400> 191
atataaatta gactaagtgt tttcaaataa atctaaatct tcagcatgat gtgttgtgta 60
taattggagt agatattaat taagtcccct gtataatgtt ttgtaatttt gcaaaacata 120
tcttgagttg tttaaacagt caaaatgttt gatattttat accagcttat gagctcaaag 180
tactacagca aagcctagcc tgcatatcat tcacccaaaa caaagtaata gcgcctcttt 240
tattattttg actgaatgtt ttatggaatt gaaagaaaca tacgttcttt tcaagacttc 300
ctcatgaatc tntcaattat aggaaaagtt attgtgataa aataggaaca gctgaaagat 360
tgattaatga actattgtta attcttccta ttttaatgaa tgacattgaa ctgaattttt 420
tgtctgttaa atgaacttga tagctaataa aaagncaact agccatcaaa aaaaaaaaaa 480
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
72
aaaaaaaaa 489
<210> 192
<211> 516
<212> DNA
<213> Homo Sapiens
<400> 192
acttcaaagc cagctgaagg aaagaggaag tgctagagag agcccccttc agtgtgcttc 60
tgacttttac ggacttggct tgttagaagg ctgaaagatg atggcaggaa tgaaaatcca 120
gcttgtatgc atgctactcc tggctttcag ctcctggagt ctgtgctcag attcagaaga 180
ggaaatgaaa gcattagaag cagatttctt gaccaatatg catacatcaa agattagtaa 240
agcacatgtt ccctcttgga agatgactct gctaaatgtt tgcagtcttg taaataattt 300
gaacagccca gctgaggaaa caggagaagt tcatgaagag gagcttgttg caagaaggaa 360
cttcttactg ctttagatgg ctttagcttg gaagcaatgt tgacaatata ccagctccac 420
aaaatctgtc acagcagggc ttttcaacac tgggagttaa tccaggaaga tattcttgat 480
actggaaatg acaaaaatgg aaaggaagaa gtcata 516
<210> 193
<211> 1409
<212> DNA
<213> Homo Sapiens
<400> 193
tgattctttt ccaaaacttt tagccatagg gtcttttata gacagggata gtaaaatgaa 60
aattgagaaa tataagatga aaaggaatgg taaaaatatc ttttaggggg cttttaattg 120
gtgatctgaa atcttgggag aagctgttct tttcaggcct gaggtgctct tgactgtcgc 180
ctgcgcactg tgtaccccga gcaacattct aagggtgtgc tttcgccttg gctaactcct 240
ttgacctcat tcttcatata gtagtctagg aaaaagttgc aggtaattta aactgtctag 300
tggtacatag taactgaatt tctattccta tgagaaatga gaattattta tttgccatca 360
acacatttta tactttgcat ctccaaattt attgcggcga gacttgtcca ttgtgaaagt 420
tagagaacat tatgtttgta tcatttcttt cataaaacct caagagcatt tttaagccct 480
tttcatcaga cccagtgaaa actaaggata gatgtttttt aactggaggt ctcctgataa 540
ggagaacaca atccaccatt gtcatttaag taataagaca ggaaattgac cttgacgctt 600
tcttgttaaa tagatttaac aggaacatct gcacatcttt tttccttgtg cactatttgt 660
ttaattgcag tggattaata cagcaagagt gccacattat aactaggcaa ttatccattc 720
ttcaagactt agttattgtc acactaattg atcgtttaag gcataagatg gtctagcatt 780
aggaacatgt gaagctaatc tgctcaaaaa gatcaacaaa ttaatattgt tgctgatatt 840
tgcataattg gctgcaatta tttaatgttt aattgggttg atcaaatgag attcagcaat 900
tcacaagtgc attaatataa acagaactgg ggcacttaaa atgataatga ttaacttata 960
ttgcatgttc tcttcctttc acttttttca gtgtctacat ttcagaccga gtttgtcagc 1020
ttttttgaaa acacatcagt agaaaccaag attttaaaat gaagtgtcaa gacgaaggca 1080
aaacctgagc agttcctaaa aagatttgct gttagaaatt ttctttgtgg cagtcattta 1140
ttaaggattc aactcgtgat acaccaaaag aagagttgac ttcagagatg tgttccatgc 1200
tctctagcac aggaatgaat aaatttataa cacctgcttt agcctttgtt ttcaaaagca 1260
caaaggaaaa gtgaaaggga aagagaaaca agtgactgag aagtcttgtt aaggaatcag 1320
gttttttcta cctggtaaac attctctatt cttttctcaa aagattgttg taagaaaaaa 1380
tgtaagmcaa aaaaaaaaaa aaaaaaaaa 1409
<210> 194
<211> 441
<212> DNA
<213> Homo Sapiens
<400> 194
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
73
cagatttcgg tagccatctc cctccaaata tgtctctttc tgctttctta gtgcccatta 60
tttccccttc tcctttcttc tgtcactgcc atctccttct tggtcttccc attgttcttt 120
aactggccgt aatgtggaat tgatatttac attttgatac ggtttttttc ttggcctgtg 180
tacgggattg cctcatttcc tgctctgaat tttaaaatta gatattaaag ctgtcatatg 240
gtttcctcac aaaagtcaac aaagtccaaa caaaaatagt ttgccgtttt actttcatcc 300
attgaaaaag gaaattgtgc ctcttgcagc ctaggcaaag gacatttagt actatcgatt 360
ctttccaccc tcacgatgac ttgcggttct ctctgtagaa aagggatggc ctaagaaata 420
caactaaaaa aaaaaaaaaa a 441
<210> 195
<211> 707
<212> DNA
<213> Homo Sapiens
<400> 195
cagaaaaata tttggaaaaa atataccact tcatagctaa gtcttacaga gaagaggatt 60
tgctaataaa acttaagttt tgaaaattaa gatgcaggta gagcttctga actaatgccc 120
acagctccaa ggaagacatg tcctatttag ttattcaaat acaagttgag ggcattgtga 180
ttaagcaaac aatatatttg ttagaacttt gtttttaaat tactgttcct tgacattact 240
tataaagagt ctctaacttt cgatttctaa aactatgtaa tacaaaagta tagtttcccc 300
atttgataaa aggccaatga tactgagtag gatatatgcg tatcatgcta cttcattcag 360
tgtgtctgtt tttaatacta ataaggcagt ttgacagaaa ttatttcttt gggactaagg 420
tgattatcat ttttttcccc ttcaaaattg tgctttaagt gctgataacc acaggcagat 480
tgcaaagaac tgataaggca acaaaagtag agaattttag gatcaaaggc atgtaactga 540
aaggtaacaa cagtacataa gcgacaactg gggaaggcag cagtgaaaca tgtttgtggg 600
gttaagtgag tcattgtaaa taaggaattt gcacatttat tttctgtcga cgcggccgcc 660
actgtgctgg atatctgcag aattccacca cactggacta gtggatc 707
<210> 196
<211> 552
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<222> (1). .(552)
<223> n = A,T,C or G
<400> 196
tggccagcca gcctgatgtg gatggcttcc ttggggtggt gcttccctca agcccgaatt 60
ngtggacatc atcaatgcca aacaatgagc cccatccatt ttccctaccc ttcctgccaa 120
gccagggant aagcagccca gaagcccagt aactgccctt tccctgcata tgcttttgat 180
ggtgtcatnt gctccttcct gtggcctcat ccaaactgta tnttccttta ctgtttatat 240
nttcaccctg taatggttgg gaccaggcca atcccttntc cacttactat aatggttgga 300
actaaacgtc accaaggtgg cttntccttg gctgaganat ggaaggcgtg gtgggatttg 360
ctnctgggtt ccctaggccc tagtgagggc agaagagaaa ccatcctntc ccttnttaca 420
ccgtgaggcc aagatcccct cagaaggcag gagtgctgcc ctntcccatg gtgcccgtgc 480
ctntgtgctg tgtatgtgaa ccacccatgt gagggaataa acctggcact aggaaaaaaa 540
aaaaaaaaaa as 552
<210> 197
<211> 449
<212> DNA
<213> Homo Sapiens
CA 02384499 2002-03-08
WO 01/18046 PCT/US00/24827
74
<220>
<221> misc_feature
<222> (1). .(449)
<223> n = A,T,C or G
<400> 197
ctccagagac aacttcgcgg tgtggtgaac tctctgagga aaaacacgtg cgtggnanca 60
agtgactgag acctanaaat ccaagcgttg gaggtcctga ggccagccta agtcgcttca 120
aaatggaacg aaggcgtttg cggggttcca ttcagagccg atacatcagc atgagtgtgt 180
ggacaagccc acggagactt gtggagctgg cagggcagag cctgctgaag gatgaggccc 240
tggccattgc ccgccctgga gttgctgccc agggagctct tcccgccact cttcatggca 300
gcctttgacg ggagacacag ccagaccctg aaggcaatgg tgcaggcctg gcccttcacc 360
tgcctccctc tgggagtgct gatgaaggga caacatcttc acctggagac cttcaaagct 420
gtgcttgatg gacttgatgt gctccttgc 449
<210> 198
<211> 606
<212> DNA
<213> Homo Sapiens
<400> 198
tgagtttgcc cccttacccc catcccagtg aatatttgca attcctaaag acgtgttttg 60
attgtcacac ctgggtgggg aacatgctac tggcatctaa tgcatagagg gcagtaatgc 120
tgctaaacat ctttcaacgc acaggacaga gccccacaaa agagaattat ctagccccaa 180
atgtccataa cactgctgtt gagaaaacct accgcaggat cttactgggc ttcataggta 240
agcttgcctt tgttctggct tctgtagata tataaaataa agacactgcc cagtccctcc 300
ctcaacgtcc cgagccaggg ctcaaggcaa ttccaataac agtagaatga acactaaata 360
ttgatttcaa aatctcagca actagaagaa tgaccaacca tcctggttgg cctgggactg 420
tcctagtttt agcattgaaa gtttcaggtt ccaggaaagc cctcaggcct gggctgctgg 480
tcaccctagc agctgaggga ctcttcaata cagaattagt ctttgtgcac tggagatgaa 540
tatactttaa tttgtaacat gtgaaaacat ctataaacat ctactgaagc ctgttcttgt 600
ctgcac 606
<210> 199
<211> 369
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<222> (1). .(369)
<223> n = A,T,C or G
<400> 199
ggcaactttt tgcggattgt tcttgcttnc aggctttgcg ctgcaaatcc agtgctacca 60
gtgtgaagaa ttccagctga acaacgactg ctcctccccc gagttcattg tgaattgcac 120
ggtgaacgtt caagacatgt gtcagaaaga agtgatggag caaagtgccg ggatcatgta 180
ccgcaagtcc tgtgcatcat cagcggcctg tctcatcgcc tctgccgggt accagtcctt 240
ctgctcccca gggaaactga actcagtttg catcagctgc tgcaacaccc ctctttgtaa 300
cgggccaagg cccaagaaaa ggggaagttc tgcctcggcc ctcangccat ggctccgcac 360
caccatcct 369
